Pathogenic biosafety is a worldwide concern. Tools for analyzing pathogenic biosafety, that are precise, rapid and field-deployable, are highly demanded. Recently developed biotechnological tools, especially those utilizing CRISPR/Cas systems which can couple with nanotechnologies, have enormous potential to achieve point-of-care (POC) testing for pathogen infection. In this review, we first introduce the working principle of class II CRISPR/Cas system for detecting nucleic acid and non-nucleic acid biomarkers, and highlight the molecular assays that leverage CRISPR technologies for POC detection. We summarize the application of CRISPR tools in detecting pathogens, including pathogenic bacteria, viruses, fungi and parasites and their variants, and highlight the profiling of pathogens' genotypes or phenotypes, such as the viability, and drug-resistance. In addition, we discuss the challenges and opportunities of CRISPR-based biosensors in pathogenic biosafety analysis.

Droplet microfluidic technology has revolutionized biomolecular analytical research, as it has the capability to reserve the genotype-to-phenotype linkage and assist for revealing the heterogeneity. Massive and uniform picolitre droplets feature dividing solution to the level that single cell and single molecule in each droplet can be visualized, barcoded, and analyzed. Then, the droplet assays can unfold intensive genomic data, offer high sensitivity, and screen and sort from a large number of combinations or phenotypes. Based on these unique advantages, this review focuses on up-to-date research concerning diverse screening applications utilizing droplet microfluidic technology. The emerging progress of droplet microfluidic technology is first introduced, including efficient and scaling-up in droplets encapsulation, and prevalent batch operations. Then the new implementations of droplet-based digital detection assays and single-cell muti-omics sequencing are briefly examined, along with related applications such as drug susceptibility testing, multiplexing for cancer subtype identification, interactions of virus-to-host, and multimodal and spatiotemporal analysis. Meanwhile, we specialize in droplet-based large-scale combinational screening regarding desired phenotypes, with an emphasis on sorting for immune cells, antibodies, enzymatic properties, and proteins produced by directed evolution methods. Finally, some challenges, deployment and future perspective of droplet microfluidics technology in practice are also discussed.

With the progressive nanofabrication technology, plasmonic nanoparticles (PNPs) have been increasingly deployed in the field of biosensing. PNPs have favorable biocompatibility, conductivity, and tunable optical properties. In addition, the localized surface plasmon resonance (LSPR) of PNPs plays a vital role in surface-enhanced Raman scattering (SERS). PNPs-based SERS biosensing enables wide-ranging applications for sensitive detection and high spatial and temporal resolution imaging. Numerous reviews of PNPs in the field of SERS biosensing highlight the fabrication or applications in one or more fields. However, the specific strategies for the SERS biosensor construction had not been summarized systematically. Thus, this work offers a comprehensive overview of SERS enhancement strategies based on PNPs, with a focus on SERS label-free detection along with label detection sensing construction, as well as its challenges and future trends.

Imperceptible wireless wearable devices are critical to advance digital medicine with the goal to capture clinical-grade biosignals continuously. Design of these systems is complex because of unique interdependent electromagnetic, mechanic and system level considerations that directly influence performance. Typically, approaches consider body location, related mechanical loads, and desired sensing capabilities, however, design for real world application context is not formulated. Wireless power casting eliminates user interaction and the need to recharge batteries, however, implementation is challenging because the use case influences performance. To facilitate a data-driven approach to design, we demonstrate a method for personalized, context-aware antenna, rectifier and wireless electronics design that considers human behavioral patterns and physiology to optimize electromagnetic and mechanical features for best performance across an average day of the target user group. Implementation of these methods result in devices that enable continuous recording of high-fidelity biosignals over weeks without the need for human interaction.

Fish health/quality issues are increasingly attracting attention during waterless and low-temperature transportation. Nondestructive detection has become a great need for an effective method to improve fish health/quality. Currently, emerging Internet of Things, novel flexible electronics and data fusion technology have received great interest for nondestructive detection on live fish health/quality. This paper analysized nondestructive detection mechanisms using novel flexible sensing technology to achieve high-precision sensing of key parameters, and machine learning based data fusion modeling to achieve live fish health/quality nondestructive evaluation during waterless and low-temperature transportation. Recent studies on novel flexible electrochemical and physiological biosensors development and application for solving key ambient and physiological parameter sensing were summarized. The ML based data fusion modeling framework and application for live fish health/quality nondestructive evaluation was also highlighted. The future perspective is also proposed to provide promising solutions for accurate sensing of multi-parameter and real applications of live fish health/quality nondestructive detection during waterless and low-temperature transportation.

Organoids are emerging in vitro models of human physiology. Neural models require the evaluation of functional activity of single cells and networks, which is commonly measured by microelectrode arrays. The characteristics of organoids clash with existing in vitro or in vivo microelectrode arrays. With inspiration from implantable mesh electronics and growth of organoids on polymer scaffolds, we fabricated suspended hammock-like mesh microelectrode arrays for neural organoids. We have demonstrated the growth of organoids enveloping these meshes and the culture of organoids on meshes for up to one year. Furthermore, we present proof-of-principle recordings of spontaneous electrical activity across the volume of an organoid. Our concept enables a new class of microelectrode arrays for in vitro models of three-dimensional electrically active tissue.

Researchers have struggled to develop highly reliable and sensitive surface-enhanced Raman scattering (SERS) substrates for detecting compounds in complicated systems. In this work, a strategy by constructing Au cores with incompletely wrapped Prussian blue (PB) for highly reliable and sensitive SERS substrate is proposed. The wrapped PB layers can provide the internal standard (IS) to calibrate the SERS signal floatation, whereas the exposed surface of Au cores offers the enhancement effect. The balance between the signal self-calibration and enhancement (hence the trade-off between SERS reliability and sensitivity) is obtained by the approximate semi-wrapping configuration of PB layers on Au cores (i.e., SW-Au@PB). The proposed SW-Au@PB nanoparticles (NPs) exhibit the similar enhancement factor as the pristine Au NPs and contribute to the ultralow RSD (8.55%) of calibrated SERS signals using R6G as probe molecules. The simultaneously realized reliability and sensitivity of SW-Au@PB NPs also enables the detection of hazardous pesticide residues such as paraquat and thiram in herbal plants, with the average detection accuracy up to 92%. Overall, this work mainly provides a controllable synthetic strategy for incompletely wrapped NPs, and most importantly, explores the potential with a proof-of-concept practical application in accurate and sensitive Raman detection of hazardous substances with varying solubility.

In this work, we report a versatile and tunable platform for the construction of various cell array biochips using a simple soft lithographic approach to pattern polydopamine (PDA) arrays via microcontact printing (μCP). Instead of direct polymerization of PDA on the polydimethylsiloxane (PDMS) tips, dopamine monomers were first printed on the substrate followed by a self-oxidative polymerization step facilitated by ammonia vapor to grow PDA in situ, which greatly reduced the reaction time and prevented the PDMS tips from damaging. The improved robustness and utility of the PDMS tips allows the formation of tunable PDA array chips with controllable PDA feature size and shape. As a result, single cell, multi-cells and cell line arrays can be constructed. The obtained cell array chips showed high single cell capture efficiency, providing a standardized single cell array analysis platform. Meanwhile, the adhered cells can maintain excellent viability and proliferation ability on the PDA chips. Moreover, a cytotoxicity sensor with single cell resolution was enabled on the single cell array chip. This work provides a promising cell array biochip platform for high-throughput cellular analysis and cell screening.

Antibody profiling is a fundamental component of understanding the humoral response in a wide range of disease areas. Most currently used approaches operate by capturing antibodies onto functionalised surfaces. Such measurements of surface binding are governed by an overall antibody titre, while the two fundamental molecular parameters, antibody affinity and antibody concentration, are challenging to determine individually from such approaches. Here, by applying microfluidic diffusional sizing (MDS), we show how we can overcome this challenge and demonstrate reliable quantification of alloantibody binding affinity and concentration of alloantibodies binding to Human Leukocyte Antigens (HLA), an extensively used clinical biomarker in organ transplantation, both in buffer and in crude human serum. Capitalising on the ability to vary both serum and HLA concentrations during MDS, we show that both affinity and concentration of HLA-specific antibodies can be determined directly in serum when neither of these parameters is known. Finally, we provide proof of principle in clinical transplant patient sera that our assay enables differentiation of alloantibody reactivity against HLA proteins of highly similar structure, providing information not attainable through currently available techniques. These results outline a path towards detection and in-depth profiling of humoral immunity and may enable further insights into the clinical relevance of antibody reactivity in clinical transplantation and beyond.

In situ acquisition of spatial distribution of biochemical substances is important in cell analysis, cancer detection and other fields. Optical fiber biosensors can achieve label-free, fast and accurate measurements. However, current optical fiber biosensors only acquire single-point of biochemical substance content. In this paper, we present a distributed optical fiber biosensor based on tapered fiber in optical frequency domain reflectometry (OFDR) for the first time. To enhance evanescent field at a relative long sensing range, we fabricate a tapered fiber with a taper waist diameter of 6 μm and a total stretching length of 140 mm. Then the human IgG layer is coated on the entire tapered region by polydopamine (PDA) -assisted immobilization as the sensing element to achieve to sense anti-human IgG. We measure shifts of the local Rayleigh backscattering spectra (RBS) caused by the refractive index (RI) change of an external medium surrounding a tapered fiber after immunoaffinity interactions by using OFDR. The measurable concentration of anti-human IgG and RBS shift has an excellent linearity in a range from 0 ng/ml to 14 ng/ml with an effective sensing range of 50 mm. The concentration measurement limit of the proposed distributed biosensor is 2 ng/ml for anti-human IgG. Distributed biosensing based on OFDR can locate a concentration change of anti-human IgG with an ultra-high sensing spatial resolution of 680 μm. The proposed sensor has a potential to realize a micron-level localization of biochemical substances such as cancer cells, which will open a door to transform single-point biosensor to distributed biosensor.

Ready-to-use in vitro diagnosis of multiple genotypes is vital for the prevention and treatment of cervical cancer. Herein, a paper-film-based interactive dual readout differential lateral flow biosensor is proposed to simultaneously assay two high-risk types of human papillomavirus (HPV) within the body-fluid. The CuCo2S4/ZnIn2S4 heterostructure is fabricated on the paper-film compound chip with high thermostability, and surface sulfur vacancy is introduced by mild annealing treatment to endow unexceptionable photoexcitation activity, such structure can be served as an initial energy harvester and converter. With the assistance of differential channels, the dual-target-propelled self-assembly of annular DNA and the cleavage activity of CRISPR-Cas12a are stepwise activated by sequential solution transfer. Accordingly, the input and release of polydopamine-coated gold nanoparticles with photothermal/photoelectric characteristic were implemented. The fabricated biosensor not only realized intelligent thermal-response without large instruments, but also actuated dynamic interfacial charge separation and transfer kinetics to further transmit photoelectric-signal, resulting in desirable interactive dual-signal with low limit-of-detection (0.21 pM for HPV-18 and 42.92 pM for HPV-16). Thanks to the sophisticated design of differential lateral flow paper-film compound chip and interactive dual-signal amplification strategy, sensitive detection of two HPV genotypes is realized, which provides a promising candidate for home medical intelligent diagnosis.

An accurate and comprehensive assessment of platelet function is essential for managing patients who receive antiplatelet therapies or require platelet transfusion either for treating active bleeding or for prophylaxis. Platelets contribute to clotting by undergoing a series of highly regulated functional responses including adhesion, spreading, granular secretion, aggregation, and cytoskeletal contraction. However, current platelet function assays evaluate only partial aspects of this intricate process and often under non-physiological testing conditions. Herein, we describe the development of a new approach to measure multiple key platelet function-related parameters, in a more physiologically relevant ex vivo semi-rigid microenvironment using a membrane capacitance sensor (MCS). MCS response to clotting provided three sensing parameters with sensitivities towards platelet counts, stimulation strengths, and activation pathways. Live confocal fluorescent imaging of stimulated platelets on MCS suggests that the presented system can readily and accurately convert the dynamics of cytoskeletal reorganization into analyzable electrical signals. Together, this new completely electrical sensing platform can be a promising diagnostic venue to recognize the impairment of primary hemostatic functions, evaluate the efficacy of therapeutic interventions, and gain further insights into the mechanisms of platelets in hemostasis and thrombosis.

Prevention of food spoilage, environmental bio-contamination, and pathogenic infections requires rapid and sensitive bacterial detection systems. Among microbial communities, the bacterial strain of Escherichia coli is most widespread, with pathogenic and non-pathogenic strains being biomarkers of bacterial contamination. Here, we have developed a fM-sensitive, simple, and robust electrocatalytically-amplified assay facilitating specific detection of E.coli 23S ribosomal rRNA, in the total RNA sample, after its site-specific cleavage by RNase H enzyme. Gold screen-printed electrodes (SPE) were electrochemically pre-treated to be productively modified with a methylene-blue (MB) - labelled hairpin DNA probes, which hybridization with the E. coli-specific DNA placed MB in the top region of the DNA duplex. The formed duplex acted as an electrical wire, mediating electron transfer from the gold electrode to the DNA-intercalated MB, and further to ferricyanide in solution, enabling its electrocatalytic reduction otherwise impeded on the hairpin-modified SPEs. The assay facilitated 20 min 1 fM detection of both synthetic E. coli DNA and 23S rRNA isolated from E.coli (equivalent to 15 CFU mL-1), and can be extended to fM analysis of nucleic acids isolated from any other bacteria.

Circular RNAs (circRNAs) can act as molecular sponges of microRNA (miRNA) to form circRNA-miRNA axis, which regulates the expressions of downstream proteins. Although the mechanism has been widely reported in various bioprocesses, there is still a lack of reliable and facile way to intuitively monitor and locate the interaction between circRNA and miRNA inside living cells. In this study, multiple DNA probes are designed and loaded onto two-dimensional Cu metal-organic framework (2D Cu-MOF) nanosheets for one-step analysis of circRNA-miRNA axis. The nanosheets serve as not only powerful fluorescence quenchers but also excellent nanocarriers of abundant DNA probes for further assembly. The Probes@Cu-MOF complex can be applied to track the circRNA-miRNA axis in living cells with high sensitivity and co-localization analysis. This platform combines the transmembrane advantage of nanosheets and the signal amplification ability of localized DNA cascade assembly, so it holds great potential for understanding the biological functions of circRNA-miRNAs in cancer pathogenesis and for therapeutic applications.

β-myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite.

In this work, a novel ECL biosensor has been developed based on bimetallic MXene derivative QDs (Mo2TiC2 QDs) and SnS2 nanosheets/lipid bilayer to detect the gastric cancer marker miRNA-27a-3p. On the one hand, the inter-band excitation of Mo2TiC2 QDs can inject the additional carriers, which were less suppressed by boundary effects and made a significant contribution to the luminescence process. Semiconductor Mo2TiC2 further inhibited the formation of internal electric field and potential oxidation. Therefore, Mo2TiC2 QDs processed superior luminous intensity and better stability. On the other hand, SnS2 nanosheets coated with phospholipid bilayer were designed as sensing interface. SnS2 nanosheets not only enhanced the luminous intensity of Mo2TiC2 QDs by virtue of their large surface area and low dielectric constant, but also improved the stability of lipid bilayer. Due to the excellent properties and synergy work of Mo2TiC2 QDs and the lipid bilayer-modified SnS2 nanosheets, the sensing system displayed high sensitivity and good reproducibility in the analysis application. As a result, the biosensor displayed good linear correlation between the ECL intensity and the concentration of miRNA-27a-3p over a wide range from 1 fM to 10 nM with the detection limit as low as 1 fM. The sensing system including the joint contribution of Mo2TiC2 QDs, SnS2 nanosheets and lipid bilayers had great potential for clinical applications.

There is a growing but unmet need for point-of-care detection of prostate-specific antigen (PSA) in body fluid which may facilitate early diagnosis and therapy of prostate cancer in a cost-effective and user-friendly way. Low sensitivity and narrow detection range limits applications of point-of-care testing in practice. Here, an immunosensor is first presented based on shrink polymer and integrated into a miniaturized electrochemical platform for detecting PSA in clinical samples. The sensing electrode was prepared by sputtering a gold film on shrink polymer, followed by heating to shrink the electrode to a small size with wrinkles from nano-scale to micro-scale. These wrinkles can be directly regulated by the thickness of the gold film with high specific areas for enhancement of antigen-antibody binding (3.9 times). A distinct difference between electrochemical active surface area (EASA) and response to PSA of shrink electrodes was observed and discussed. The electrode was treated with air plasma and modified with self-assembled graphene to further enhance the sensor's sensitivity (10.4 times). The shrink sensor with gold 200 nm thick integrated into the portable system was validated by a label-free immunoassay for detection of PSA in 20 μL serum within 35 mins. It exhibited a limit of detection of 0.38 fg/mL, the lowest among label-free PSA sensors, and a wide linear response from 10 fg/mL to 1000 ng/mL. Moreover, the sensor demonstrated reliable assay results in clinical serums, comparable to the commercial chemiluminescence instrument, confirming its feasibility for clinical diagnosis.

Typhoid fever is an acute illness caused by Salmonella Typhi and the current diagnostic gap leads to inaccurate, over-diagnosis of typhoid leading to excessive use of antibiotics. Herein, to address the challenges we describe a new rapid color-shift assay based on a novel bifunctional nanobioprobe (Vi-AgNP probe) that is functionalized with specific biomarker Vi polysaccharide and also has the co-presence of Ag as urease inhibitor. The immunoreactions between the Vi with specific antibodies (Abs) present in typhoid patient sample forms a shielding barrier over Vi-AgNP probe rendering the urease to be active, generating colored output. Vi polysaccharide coating on the AgNP was visualized using HRTEM. TEM was performed to get insight into shielding barrier formation by the Abs. MST (microscale thermophoresis) data showed less binding Kd of 7.43 μM in presence of Abs whereas probe with urease showed efficient binding with Kd 437 nM. The assay was validated using 53 human sera samples and proven effective with 100% sensitivity. The assay showed relative standard deviation (RSD) of 4.3% estimated using rabbit anti-Vi Abs. The entire procedure could be completed within 15 min. Unlike lateral flow based assays, our assay does not require multiple combination of Abs for detection. The assay format was also found compatible in paper strip test that provides promising opportunities to develop low-cost on-spot assay for clinical diagnostics.

Due to the extended culture period and various inconveniences in vitro culture, the detection of invasive fungi is rather difficult, leading to high mortality rates of the diseases caused by them. It is, however, crucial for clinical therapy and lowering patient mortality to quickly identify invasive fungus from clinical specimens. A promising non-destructive method for finding fungi is surface-enhanced Raman scattering (SERS), however, its substrate has a low level of selectivity. Clinical sample components can obstruct the target fungi's SERS signal on account of their complexity. Herein, an MNP@PNIPAMAA hybrid organic-inorganic nano-catcher was created by using ultrasonic-initiated polymerization. The caspofungin (CAS), a fungus cell wall-targeting drug, is used in this study. We investigated MNP@PNIPAMAA-CAS as a technique to rapidly extract fungus from complex samples under 3 s. SERS could subsequently be used to instantly identify the fungi that were successfully isolated with an efficacy rate of about 75%. The entire process took just 10 min. This method is an important breakthrough that might be advantageous in terms of the rapid detection of invasive fungi.

The literature on voltammetry and electrochemical impedance spectroscopy (EIS) recognises the importance of using large-amplitude sinusoidal perturbations to better characterise electrochemical systems. To identify the parameters of a given reaction, various electrochemical models with different sets of values are simulated and compared against the experimental data to determine the best-fit set of parameters. However, the process of solving these nonlinear models is computationally expensive. This paper proposes analogue circuit elements for synthesising surface-confined electrochemical kinetics at the electrode interface. The resultant analogue model could be used as a solver to compute reaction parameters as well as a tracker for ideal biosensor behaviour. The performance of the analogue model was verified against numerical solutions to theoretical and experimental electrochemical models. Results show that the proposed analogue model has a high accuracy of at least 97% and a wide bandwidth of up to 2 kHz. The circuit consumed an average power of 9 μW.

In this study, we aimed to introduce a new electrochemical aptasensor based on the tyramide signal amplification (TSA) technology for a highly-sensitive detection of the pathogenic bacterium, Staphylococcus aureus, as a model of foodborne pathogens. In this aptasensor, the primary aptamer, SA37, was used to specifically capture bacterial cells; the secondary aptamer, SA81@HRP, was used as the catalytic probe; and a TSA-based signal enhancement system comprising of biotinyl-tyramide and streptavidin-HRP as electrocatalytic signal tags was adopted to fabricate the sensor and improve the detection sensitivity. S. aureus cells were selected as the pathogenic bacteria to verify the analytical performance of this TSA-based signal-enhancement electrochemical aptasensor platform. After the simultaneous binding of SA37-S. aureus-SA81@HRP formed on the gold electrode, thousands of @HRP molecules could be bound onto the biotynyl tyramide (TB) displayed on the bacterial cell surface through a catalytic reaction between HRP and H2O2, resulting in the generation of the highly amplified signals mediated by HRP reactions. This developed aptasensor could detect S. aureus bacterial cells at an ultra-low concentration, with a limit of detection (LOD) of 3 CFU/mL in buffer. Furthermore, this chronoamperometry aptasensor successfully detected target cells in both tap water and beef broth with LOD to be 8 CFU/mL, which are very high sensitivity and specificity. Overall, this electrochemical aptasensor using TSA-based signal-enhancement could be a very useful tool for the ultrasensitive detection of foodborne pathogens in food and water safety and environmental monitoring.

The high attrition rate of drug candidates contributes to the long duration and high cost in modern drug development. A major barrier in drug development is the poor predicting power of the preclinical models. In the current study, a human pulmonary fibrosis on chip system was developed for the preclinical evaluation of anti-fibrosis drugs. Pulmonary fibrosis is a severe disease characterized by progressive tissue stiffening that leads to respiration failure. To recapitulate the unique biomechanical feature of the fibrotic tissues, we developed flexible micropillars that can serve as in-situ force sensors to detect the changes in the mechanical properties of engineered lung microtissues. Using this system, we modeled the fibrogenesis of the alveolar tissues including the tissue stiffening and the expression of α-smooth muscle actin (α-SMA) and pro-collagen. Two anti-fibrosis drug candidates that are currently under clinical trials (KD025 and BMS-986020) were tested for their potential anti-fibrosis efficacy and the results were compared to those of FDA-approved anti-fibrosis drugs pirfenidone and nintedanib. Both pre-approval drugs were effective in inhibiting transforming growth factor beta 1 (TGF-β1) induced increases in tissue contractile force, stiffness and expressions of fibrotic biomarkers, which are similar to the effects of FDA-approved anti-fibrosis drugs. These results demonstrated the potential utility of the force-sensing fibrosis on chip system in the pre-clinical development of anti-fibrosis drugs.

The low polarization and low Raman cross section characteristics of glutathione (GSH) make it challenging to directly detect GSH molecules through surface enhanced Raman scattering (SERS) technology. Development of nanostructures for indirect detection of GSH applied to the SERS platform is of great interest. Herein, silver nanoparticles (Ag NPs)/copper-polyphenol colloidal spheres (denoted as CuTA@Ag) with adjustable Ag NPs coverage are prepared by deposition of Ag NPs on the metal-polyphenol colloidal spheres via an interfacial polyphenol reduction method. The size and density of the Ag NPs deposited on the out layer can be readily adjusted by tailoring the concentrations of silver precursor. It leads to activity difference for the nanozyme and SERS characteristics. The SERS properties of the obtained CuTA@Ag are studied using oxTMB, catalytic products of nanozyme, as the probing molecules. They provide satisfactory SERS performance with a low detection limit of 10-7 M (S/N = 3) and linear determination in the 1-100 μM range for GSH. Moreover, it is further able to detect the glutathione content in cancer cells with well accurate and reproducible capability, catching the signs of rising cancer marker levels. This work proposes structurally tunable nanomaterials platform for a catalytic-based SERS assay, which is expected to utilize the high sensitivity of SERS tool for GSH detection in the cellular environment.

Quantum dots (QDs) have become promising electrochemiluminescence (ECL) emitters with high quantum yield and size-tunable luminescence. However, most QDs generate strong ECL emission at the cathode, developing anodic ECL-emitting QDs with excellent performance is challenging. In this work, low-toxic quaternary AgInZnS QDs synthesized by a one-step aqueous phase method were used as novel anodic ECL emitters. AgInZnS QDs exhibited strong and stable ECL emission and a low excitation potential, which could avoid the side reaction of oxygen evolution. Furthermore, AgInZnS QDs displayed high ECL efficiency (ΦECL) of 5.84, taking the ΦECL of Ru(bpy)32+/tripropylamine (TPrA) ECL system as 1. Compared to AgInS2 QDs without Zn doping and traditional anode luminescent CdTe QDs, the ECL intensity of AgInZnS QDs was 1.62 times and 3.64 times higher than that of AgInS2 QDs and CdTe QDs, respectively. As a proof-of-concept, we further designed an "on-off-on" ECL biosensor for detecting microRNA-141 based on a dual isothermal enzyme-free strand displacement reaction (SDR), which not only to achieve the cyclic amplification of the target and ECL signal, but also to construct a switch of the biosensor. The ECL biosensor had a wide linear range from 100 aM to 10 nM with a low detection limit of 33.3 aM. Together, the constructed ECL sensing platform is a promising tool for rapid and accurate diagnosis of clinical diseases.

Unwelcomed biofilms are problematic in food industries, surgical devices, marine applications, and wastewater treatment plants, essentially everywhere where there is moisture. Very recently, label-free advanced sensors such as localized and extended surface plasmon resonance (SPR) have been explored as tools for monitoring biofilm formation. However, conventional noble metal SPR substrates suffer from low penetration depth (100-300 nm) into the dielectric medium above the surface, preventing the reliable detection of large entities of single or multi-layered cell assemblies like biofilms which can grow up to a few micrometers or more. In this study, we propose using a plasmonic insulator-metal-insulator (IMI) structure (SiO2-Ag-SiO2) with a higher penetration depth based on a diverging beam single wavelength format of Kretschmann configuration in a portable SPR device. An SPR line detection algorithm for locating the reflectance minimum of the device helps to view changes in refractive index and accumulation of the biofilm in real-time down to 10-7 RIU precision. The optimized IMI structure exhibits strong penetration dependence on wavelength and incidence angle. Within the plasmonic resonance, different angles penetrate different depths, showing a maximum near the critical angle. At the wavelength of 635 nm, a high penetration depth of more than 4 μm was obtained. Compared to a thin gold film substrate, for which the penetration depth is only ∼200 nm, the IMI substrate provides more reliable results. The average thickness of the biofilm after 24 h of growth was found to be between 6 and 7 μm with ∼63% live cell volume, as estimated from confocal microscopic images using an image processing tool. To explain this saturation thickness, a graded index biofilm structure is proposed in which the refractive index decreases with the distance from the interface. Furthermore, when plasma-assisted degeneration of biofilms was studied in a semi-real-time format, there was almost no effect on the IMI substrate compared to the gold substrate. The growth rate over the SiO2 surface was higher than on gold, possibly due to differences between surface charge effects. On the gold, the excited plasmon generates an oscillating cloud of electrons, while for the SiO2 case, this does not happen. This methodology can be utilized to detect and characterize biofilms with better signal reliability with respect to concentration and size dependence.

In this study, the N-doped carbon dots were continuously synthesized by a facile microfluidic strategy at 90 °C, and their quantum yields reached 19.2%. The characteristics of the obtained carbon dots could be real-time monitored in order to synthesize carbon dots with specific properties. By incorporating the carbon dots into a well-established enzymatic cascade amplification system, an inner filter effect-based fluorescence immunoassay was set up for ultrasensitive detection of cefquinome residues in milk samples. The developed fluorescence immunoassay provided a low detection limit of 0.78 ng/mL, which satisfied the maximum residue limit set by authorities. The fluorescence immunoassay had an 50% inhibition concentration of 0.19 ng/mL against cefquinome and showed a good linear relationship from 0.013 ng/mL to 1.52 ng/mL. While, the average recovery values ranged from 77.8% to 107.8% in spiked milk samples, with relative standard deviations ranging from 6.8% to 10.9%. Compared with conventional methods, the microfluidic chip was more flexible on carbon dots synthesis and the developed fluorescence immunoassay was more sensitive and eco-friendlier for ultra-trace cefquinome residue analysis.

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

Flexible biomimetic sensors have encountered a bottleneck of sensitivity and durability, as the sensors must directly work within complex body fluid with ultra-trace biomarkers. In this work, a wearable electrochemical sensor on a modified silk fibroin substrate is developed using gold nanoparticles hosted into N-doped porous carbonizated silk fibroin (AuNPs@CSF) as active materials. Taking advantage of the inherent biocompatibility and flexibility of CSF, and the high stability and enzyme-like catalytic activity of AuNPs, AuNPs@CSF-based sensor exhibits durable stability and superior sensitivity to monitor H2O2 released from cancer cell (4T1) and glucose in sweat. The detection limits for H2O2 and glucose are low to be 1.88 μM and 23 μM respectively, and the sensor can be applied in succession within 30 days at room temperature. Further, physical cross-linking of polyurethane (PU) with SF well matches with the skin tissue mechanically and provides a flexible, robust and stable electrode-tissue interface. AuNPs@CSF is applied successfully for wearable electrochemical monitoring of glucose in human sweat.The present AuNPs@CSF will possess a potential application in clinical diagnosing of H2O2- or glucose-related diseases in future.

This manuscript presents the design and facile production of screen-printed arrays (SPAs) for the internally validated determination of raised levels of serum procalcitonin (PCT). The screen-printing methodology produced SPAs with six individual working electrodes that exhibit an inter-array reproducibility of 3.64% and 5.51% for the electrochemically active surface area and heterogenous electrochemical rate constant respectively. The SPAs were modified with antibodies specific for the detection of PCT through a facile methodology, where each stage simply uses droplets incubated on the surface, allowing for their mass-production. This platform was used for the detection of PCT, achieving a linear dynamic range between 1 and 10 ng mL-1 with a sensor sensitivity of 1.35 × 10-10 NIC%/ng mL-1. The SPA produced an intra- and inter-day %RSD of 4.00 and 5.05%, with a material cost of £1.14. Internally validated human serum results (3 sample measurements, 3 control) for raised levels of PCT (>2 ng mL-1) were obtained, with no interference effects seen from CRP and IL-6. This SPA platform has the potential to offer clinicians vital information to rapidly begin treatment for "query sepsis" patients while awaiting results from more lengthy remote laboratory testing methods. Analytical ranges tested make this an ideal approach for rapid testing in specific patient populations (such as neonates or critically ill patients) in which PCT ranges are inherently wider. Due to the facile modification methods, we predict this could be used for various analytes on a single array, or the array increased further to maintain the internal validation of the system.

Accurate quantitative analysis of tumor markers in a wide linear range has important practical significance towards complex clinical samples in cancer identification and monitoring of tumor development stages, but remains challenging. Herein, three-layer dumbbell-like upconversion nanoparticles NaErF4:Tm@NaYF4@NaNdF4 (labeled as UCNPs) combined with G-quadruplex (G4) DNAzyme are reported for tri-modal sensing of carcinoembryonic antigen (CEA) in a wide range using upconversion luminescence (UCL), photothermal and catalysis signal readouts. Initially, dumbbell-like UCNPs were controlled synthesized by a three-dimensional epitaxial growth strategy through tuning the concentration of Nd precursors. After surface functionalization, G4zyme-UCNPs-cDNA/Apt-MB was subsequently fabricated by biotin-streptavidin interaction and DNA hybridization. Quantitative detection of CEA was achieved by competitive interaction and magnetic separation, and the intensities of tri-modal signals (light, heat and catalysis-based chrominance) of dissociative probes are linearly related to the concentration of CEA. The results showed that the tri-modal sensing method exhibited a wide linear range (0.005-2000 ng/mL) and low limit of detection (LOD) across three models: the luminescence model (0.005-50 ng/mL, LOD = 0.910 pg/mL), the catalysis model (10-1000 ng/mL, LOD = 0.387 ng/mL), and the temperature model (50-2000 ng/mL, LOD = 1.114 ng/mL). These findings suggest that the tri-modal sensing platform is suitable for use in the analysis of a wide range of complex and diverse clinical samples.

Gallbladder carcinoma (GBC) is one of most aggressive and lethal malignancies. Early diagnosis of GBC is crucial for determining appropriate treatment and improving chances of cure. Chemotherapy represents the main therapeutic regimen for unresectable GBC patients to inhibit tumor growth & metastasis. But, chemoresistance is the major cause of GBC recurrence. Thus, there is an urgent need to explore potentially non-invasive and point-of-care approaches to screen GBC and monitor their chemoresistance. Herein, we established an electrochemical cytosensor to specifically detect circulating tumor cells (CTCs) and their chemoresistance. Trilayer of CdSe/ZnS quantum dots (QDs) were cladded upon SiO2 nanoparticles (NPs), forming Tri-QDs/PEI@SiO2 electrochemical probes. Upon conjugation of anti-ENPP1, the electrochemical probes were able to specifically label captured CTCs from GBC. The detection of CTCs and chemoresistance were realized by square wave anodic stripping voltammetric (SWASV) responses to anodic stripping current of Cd 2+ ion when cadmium in electrochemical probes was dissolved and eventually electrodeposited on bismuth film-modified glassy carbon electrode (BFE). Taking use of this cytosensor, one ensured the screening of GBC and limit of detection for CTCs approaches to ～10 cells/mL. Furthermore, by monitoring phenotypic changes of CTCs after drug treatment, the diagnosis of chemoresistance was achieved by our cytosensor.

COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an ongoing global pandemic with economic and social disruption. Moreover, the virus has persistently and rapidly evolved into novel lineages with mutations. The most effective strategy to control the pandemic is suppressing virus spread through early detection of infections. Therefore, developing a rapid, accurate, easy-to-use diagnostic platform against SARS-CoV-2 variants of concern remains necessary. Here, we developed an ultra-sensitive label-free surface-enhanced Raman scattering-based aptasensor as a countermeasure for the universal detection of SARS-CoV-2 variants of concern. In this aptasensor platform, we discovered two DNA aptamers that enable binding to SARS-CoV-2 spike protein via the Particle Display, a high-throughput screening approach. These showed high affinity that exhibited dissociation constants of 1.47 ± 0.30 nM and 1.81 ± 0.39 nM. We designed a combination with the aptamers and silver nanoforest for developing an ultra-sensitive SERS platform and achieved an attomolar (10-18 M) level detection limit with a recombinant trimeric spike protein. Furthermore, using the intrinsic properties of the aptamer signal, we demonstrated a label-free aptasensor approach, enabling use without the Raman tag. Finally, our label-free SERS-combined aptasensor succeeded in detecting SARS-CoV-2 with excellent accuracy, even in clinical samples with variants of concern, including the wild-type, delta, and omicron variants.

Sensitive and reliable detection of antibiotics is of great significance for environmental and food safety due to its high risk in trace concentrations. Herein, we developed a fluorescence sensing system for chloramphenicol (CAP) detection based on dumbbell DNA-mediated signal amplification. Two hairpin dimers (2H1 and 2H2) were employed as the building blocks to construct the sensing scaffolds. The CAP-aptamer binding in another hairpin H0 can liberate the trigger DNA, which then activates the cyclic assembly reaction between 2H1 and 2H2. The separation of FAM and BHQ in the formed product of cascaded DNA ladder yields a high fluorescence signal for CAP monitoring. Compared with the monomer hairpin assembly between H1 and H2, the dimer hairpin assembly between 2H1 and 2H2 exhibits enhanced signal amplification efficiency and reduced reaction time. The developed CAP sensor showed a wide linear range from 10 fM to 10 nM with a detection limit of 2 fM. Importantly, this sensing platform has been successfully applied to the determination of CAP in fish, milk, and water samples with satisfactory recovery and accuracy. With the advantages of high sensitivity, mix-and-read pattern, and robustness, our proposed CAP sensor can be used as a simple and routine tool for the detection of trace amounts of antibiotic residues.

Alzheimer's disease (AD) is generally diagnosed using advanced imaging, but recent research suggests early screening using biomarkers in peripheral blood is feasible; among them, plasma tau proteins phosphorylated at threonine 231, threonine 181, and threonine 217 (p-tau217) are potential targets. A recent study indicates that the p-tau217 protein is the most efficacious biomarker. However, a clinical study found a pg/ml threshold for AD screening beyond standard detection methods. A biosensor with high sensitivity and specificity p-tau217 detection has not yet been reported. In this study, we developed a label-free solution-gated field effect transistor (SGFET)-based biosensor featuring a graphene oxide/graphene (GO/G) layered composite. The top layer of bilayer graphene grown using chemical vapor deposition was functionalized with oxidative groups serving as active sites for forming covalent bonds with the biorecognition element (antibodies); the bottom G could act as a transducer to respond to the attachment of the target analytes onto the top GO conjugated with the biorecognition element via π-π interactions between the GO and G layers. With this unique atomically layered G composite, we obtained a good linear electrical response in the Dirac point shift to p-tau217 protein concentrations in the range of 10 fg/ml to 100 pg/ml. The biosensor exhibited a high sensitivity of 18.6 mV/decade with a high linearity of 0.991 in phosphate-buffered saline (PBS); in human serum albumin, it showed approximately 90% of the sensitivity (16.7 mV/decade) in PBS, demonstrating high specificity. High stability of the biosensor was also displayed in this study.

Herein, we prepared a self-crosslinked conductive molecularly imprinted gel (CMIG) using cationic guar gum (CGG), chitosan (CS), β-cyclodextrin (β-CD), amaranth (AM) and multi-walled carbon nanotubes (MWCNTs) by a simple one-pot low temperature magnetic stirring method. The imine bonds, hydrogen-bonding interactions and electrostatic attractions between CGG, CS and AM facilitated CMIG gelation, while β-CD and MWCNTs enhanced the adsorption capacity and conductivity of CMIG, respectively. Next, the CMIG was deposited onto the surface of a glassy carbon electrode (GCE). After selective removal of AM, a highly sensitive and selective CMIG-based electrochemical sensor was obtained for AM determination in foods. The CMIG allowed specific recognition of AM and could also be used for signal amplification, thus improving the sensitivity and selectivity of the sensor. Due to the high viscosity and self-healing properties of the CMIG, the developed sensor was very durable retaining a 92.1% of original current after 60 consecutive measurements. Under optimal conditions, the CMIG/GCE sensor showed a good linear response for AM detection (0.02-150 μM) with a limit of detection of 0.003 μM. AM recovery tests were performed in milk powder and white vinegar samples, yielding satisfactory recoveries (89.00%-111.00%). Furthermore, the levels of AM in two kinds of carbonated drinks were analyzed with the constructed sensor and an ultraviolet spectrophotometry method, with no significant difference found of the two methods. This work demonstrates that CMIG based electrochemical sensing platforms allow the cost-effective detection of AM, with the CMIG technology likely being widely applicable to the detection of other analytes.

Circulating cell-free DNA (cfDNA) is a promising biomarker of liquid biopsy, but it still faces some difficulties in achieving sensitive and convenient detection. Herein, an Ω-shaped fiber optic localized surface plasmon resonance (FO-LSPR) biosensor based on hybridization chain reaction (HCR) coupled with gold nanoparticles (AuNPs) was developed, and applied in simple and sensitive detection of cfDNA. Specifically, one-base mismatch was designed in HCR hairpins (H1 and H2) to obtain high reaction efficiency, and AuNPs was introduced onto H1 through poly-adenine to construct HCR coupled with AuNPs strategy. Meanwhile, target cfDNA was designed into two domains: one could trigger HCR to generate dsDNA concatemer carrying numerous AuNPs, and the other could hybridize with capture DNA on the surface of Ω-shaped fiber optic (FO) probes. Thus, the presence of target cfDNA would initiate HCR, and bring the formed dsDNA concatemer and AuNPs to approach the probe surface, resulting in dramatically amplified LSPR signal. Besides, HCR required simple isothermal and enzyme-free condition, and Ω-shaped FO probe with high refractive index sensitivity just needed to be immersed into HCR solution directly for signal monitoring. Benefiting from the synergetic amplification of mismatched HCR and AuNPs, the proposed biosensor exhibited high sensitivity with a limit of detection of 14.0 pM, and therefore could provide a potential strategy for biomedical analysis and disease diagnosis.

N6-Methyladenosine (m6A) is a reversible chemical modification in eukaryotic messenger RNAs and long noncoding RNAs. The aberrant expression of RNA methyltransferase METTL3-METTL14 complex may change the m6A methylation level and cause multiple diseases including cancers. The conventional METTL3-METTL14 assays commonly suffer from time-consuming procedures and poor sensitivity. Herein, we develop a controllable amplification machinery based on MazF-activated terminal deoxynucleotidyl transferase (TdT)-assisted dendritic DNA structure assembly for ultrasensitive detection of METTL3-METTL14 complex activity in cancer cells and breast tissues. The presence of METTL3-METTL14 complex catalyzes the formation of m6A in detection probe, effectively preventing the cleavage of methylated detection probes by MazF. The methylated detection probes with 3'-OH termini can function as the primers for template-free polymerization catalyzed by TdT on magnetic beads (MBs), producing long chains of poly-thymidine (poly-T) sequences. Then poly-T sequences hybridize with signal probes that contain poly-adenine (poly-A) sequence, inducing TdT-mediated polymerization and the subsequent hybridization with more poly-A signal probes for generating dendritic DNA nanostructures assembled on MBs. After magnetic separation and elevated temperature treatment, the signal probes are disassembled from MBs to generate a high fluorescence signal. This method possesses excellent specificity and high sensitivity with a limit of detection (LOD) of 2.61 × 10-15 M, and it can accurately quantify cellular METTL3-METTL14 complex at single-cell level. Furthermore, it can screen inhibitors, evaluate kinetic parameters, and discriminate breast cancer tissues from normal tissues.

Micro-coil magnetic stimulation of brain tissue presents new challenges for MEMS micro-coil probe fabrication. The main challenges are threefold; (i) low coil resistance for high power efficiency, (ii) low leak current from the probe into the in vitro experimental set-up, (iii) adaptive MEMS process technology because of the dynamic research area, which requires agile design changes. Taking on these challenges, we present a MEMS fabrication process that has three main features; (i) multilayer resist lift-off process to pattern up to 1800-nm-thick metal films, and special care is taken to obtain high conductivity thin-films by physical vapor deposition, and (ii) all micro-coil Al wires are encapsulated in at least 200 nm of ALD alumina and 6-μm-thick parylene C such the leak resistance is high (>210 GΩ), (iii) combining a multi-step DRIE process and maskless photolithography for adaptive design and device fabrication. The entire process requires four lithography steps. Because we avoided SOI wafers and lithography mask fabrication, the design-to-device time is shortened significantly. The resulting probes are 4-mm-long, 60-μm-thick, and down to 150 μm-wide. Selected MEMS coil devices were validated in vivo using mice and compared to previous work.

Sepsis is a life-threatening immune response which is caused by a wide variety of sources and is a leading cause of mortality globally. Rapid diagnosis and appropriate antibiotic treatment are critical for successful patient outcomes; however, current molecular diagnostic techniques are time-consuming, costly and require trained personnel. Additionally, there is a lack of rapid point-of-care (POC) devices available for sepsis detection despite the urgent requirements in emergency departments and low-resource areas. Recent advances have been made toward developing a POC test for early sepsis detection that will be more rapid and accurate compared to conventional techniques. Within this context, this review discusses the use of current and novel biomarkers for early sepsis diagnosis using microfluidics devices for POC testing.

Despite non-invasive instant monitoring of sweat metabolites is becoming a general trend in early diagnostics and screening, the reliability and accuracy of the on-skin electrochemical biosensors in real-life scenarios still remain questionable. As a rule, mass transport effects in scantily excreted liquids are ignored, when considering the design of such wearable setups. Here we provide a comprehensive investigation of the disruption factors for commonly used Prussian Blue based (bio)sensors under different hydrodynamic conditions (2 × 10-5 - 5 × 100 mm s-1 electrolyte velocity). A huge effect of flow on the (bio)sensors response has been revealed and explained with transport limitations for both analyte influx and reaction product outflux. It suggests no need for improving the sensor sensitivity, while minimizing analyte consumption and enhancing product withdrawal. Some strategies concerning measurement schemes and sensor design ensuring reliable sweat analysis have been discussed and illustrated for lactate and glucose on-skin monitoring.

Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.

We report quantitative determination of extracellular H2O2 released from single COS-7 cells with high spatial resolution, using scanning electrochemical microscopy (SECM). Our strategy of depth scan imaging in vertical x-z plane was conveniently utilized to a single cell for obtaining probe approach curves (PACs) to any positions on the membrane of a live cell by simply drawing a vertical line on one depth SECM image. This SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The H2O2 concentration at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O2, and determined to be 0.020 mM by overlapping the experimental PAC with the simulated one having a known H2O2 release value. The H2O2 profile determined in this way gives insight into physiological activity of single live cells. In addition, intracellular H2O2 profile was demonstrated using confocal microscopy by labelling the cells with a luminomphore, 2',7'-dichlorodihydrofluorescein diacetate. The two methodologies have illustrated complementary experimental results of H2O2 detection, indicating that H2O2 generation is centered at endoplasmic reticula.

Implantable neural microelectrodes for recording and stimulating neural activity are critical for research in neuroscience and clinical neuroprosthetic applications. A current need exists for developing new technological solutions for obtaining highly selective and stealthy electrodes that provide reliable neural integration and maintain neuronal viability. This paper reports a novel Hollow Ring-like type electrode to sense and/or stimulate neural activity from three-dimensional neural networks. Due to its unique design, the ring electrode architecture enables easy and reliable access of the electrode to three-dimensional neural networks with reduced mechanical contact on the biological tissue, while providing improved electrical interface with cells. The Hollow Ring electrodes, particularly when coated with the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), show improved electrical properties with extremely low impedance (7 MΩ μm2) and high charge injection capabilities (15 mC/cm2), when compared to traditional planar disk-type electrodes. The ring design also serves as an optimal architecture for cell growth to create an optimal subcellular electrical-neural interface. In addition, we showed that neural signals recorded by the ring electrode were better resolved than recordings from a traditional disk-type electrode improving the signal-to-noise ratio (SNR) and the burst detection from 3D neuronal networks in vitro. Overall, our results suggest the great potential of the hollow ring design for developing next-generation microelectrodes for applications in neural interfaces used in physiological studies and neuromodulation applications.

Discrete, extended gate pH-sensitive field-effect transistors (dEGFETs) fabricated on printed circuit boards (PCBs) are a low-cost, simple to manufacture analytical technology that can be applied to a wide range of applications. Electrodeposited iridium oxide (IrOx) films have emerged as promising pH-sensitive layers owing to their theoretically high pH sensitivity and facile deposition, but typically exhibit low pH sensitivity or lack reproducibility. Moreover, to date, a combined IrOx and dEGFET PCB system has not yet been realised. In this study, we demonstrate a dEGFET pH sensor based on an extended gate manufactured on PCB that is rendered pH sensitive through an electrodeposited IrOx film, which can reliably and repeatably display beyond-Nernstian pH response. Using a combination of complementary surface analysis techniques, we show that the high pH sensitivity and repeatability of the dEGFETs are dependent on both the chemical composition and critically the uniformity of the IrOx film. The IrOx film uniformity can be enhanced through electrochemical polishing of the extended gate electrode prior to electrodeposition, leading to dEGFETs that exhibit a median pH sensitivity of 70.7 ± 5 mV/pH (n = 56) compared to only 31.3 ± 14 mV/pH (n = 31) for IrOx electrodeposited on non-polished PCB electrodes. Finally, we demonstrate the applicability of these devices by demonstrating the detection and quantification of ampicillin due to β-Lactamase enzyme activity, thus laying the foundation for cheap and ubiquitous sensors which can be applied to a range of global challenges across healthcare and environmental monitoring.

High heterogeneity in the membrane protein expression of small extracellular vesicles (sEVs) means that bulk methods relying on antibody-based capture for expression analysis have a drawback that each type of antibody may capture a different sub-population. An improved approach is to capture a representative sEV population, without any bias, and then perform a multiplexed protein expression analysis on this population. However, such a possibility has been largely limited to fluorescence-based methods. Here, we present a novel electrostatic labelling strategy and a microchip-based all-electric method for membrane protein analysis of sEVs. The method allows us to profile multiple surface proteins on the captured sEVs using alternating charge labels. It also permits the comparison of expression levels in different sEV-subtypes. The proof of concept was tested by capturing sEVs both non-specifically (unbiased) as well as via anti-CD9 capture probes (biased), and then profiling the expression levels of various surface proteins using the charge labelled antibodies. The method is the first of its kind, demonstrating an all-electrical and microchip based method that allows for unbiased analysis of sEV membrane protein expression, comparison of expression levels in different sEV subsets, and fractional estimation of different sEV sub-populations. These results were also validated in parallel using a single-sEV fluorescence technique.

Three-dimensional (3D) printed electrochemical devices are increasingly used in point-of-need and point-of-care testing. They show several advantages such as simple fabrication, low cost, fast response, and excellent selectivity and sensitivity in small sample volumes. However, there are only a few examples of analytical devices combining 3D-printed electrodes with electrochemiluminescence (ECL) detection, an electrochemical detection principle widely employed in clinical chemistry analysis. Herein, a portable, 3D-printed miniaturized ECL biosensor for glucose detection has been developed, based on the luminol/H2O2 ECL system and employing a two-electrode configuration with carbon black-doped polylactic acid (PLA) electrodes. The ECL emission is obtained by means of a 1.5V AA alkaline battery and detected using a smartphone camera, thus providing easy portability of the analytical platform. The ECL system was successfully applied for sensing H2O2 and, upon coupling the luminol/H2O2 system with the enzyme glucose oxidase, for glucose detection. The incorporation of luminol and glucose oxidase in an agarose hydrogel matrix allowed to produce ECL devices preloaded with the reagents required for the assay, so that the analysis only required sample addition. The ECL biosensor showed an excellent ability to detect glucose up to 5 mmol L-1, with a limit of detection of 60 μmol L-1. The biosensor was also used to analyse real samples (i.e., glucose saline solutions and artificial serum samples) with satisfactory results, thus suggesting its suitability for point-of-care analysis. Coupling with other oxidases could further extend the applicability of this analytical platform.

In this work, by integrating with a highly efficient multilocus domino-like cascade reaction on DNA nanonet, an ultrasensitive PEC biosensor based on dual-sensitized PDA/ZnO@MoS2 QDs photoactive material as signal probe was proposed for detection of miRNA-182-5p. The dual-sensitized PDA/ZnO@MoS2 QD composed by both of p-n and S-scheme heterojunctions on electrode generated an extremely high initial PEC signal, which however quenched by CdTe QDs decorated on DNA nanonet owing to the significant p-n quenching effect. Thereafter, the output DNA (RS) from DSN enzyme-assisted target recycling amplification triggered an ingenious multilocus domino-like DNA cascade reaction on DNA nanonet for releasing numerous CdTe QDs. Thanks to the multilocus domino-like mode that owned abundant binding sites for increasing trigger efficiency and drove cascade reaction automatically advance along four stated pathways, the target conversion rate could be improved effectively compared with that of traditional approaches, significantly enhancing the detection sensitivity. Consequently, the developed PEC biosensor exhibited a low detection limit to 0.17 fM, providing a new avenue for sensitive, fast and reliable sensing of various DNA/RNA.

Based on the admirable precision to identify target nucleic acids and the particular trans-cleavage feature, CRISPR/Cas12a system is a useful means to further improve the sensing accuracy and the design flexibility of fluorescence biosensors. However, the current construction concepts still suffer from insufficient sensitivity, unsuitable for complicated real samples and limited detection species. In this work, much efforts are achieved to address these obstacles. At first, we adopt a microsphere sustained signal enrichment, under which a home-made time-shared scanning optical tweezers assisted fluorescence imaging is employed to guarantee a stable excitation and also realize multiflux measurement. Furthermore, by taking advantage of the low background merit of the near-infrared light excited two-photon fluorescence, a commendable anti-interference capability is endowed to operate in complex media. After utilizing a functional DNA (e.g. aptamer and DNAzyme) regulated mediation pathway to respond non-nucleic acid analytes (alpha fetal protein and Pb2+), the newly-established CRISPR/Cas12a-mediated fluorescence biosensor is found to display favorable assay performance. More importantly, our analytical methodology can act as a versatile and reliable toolbox in various applications such as disease diagnosis and environmental analysis, propelling the development of CRISPR system in biosensing field.

Accurate dopamine (DA) monitoring with high stability is essential for investigating the chemical basis of brain function and pathology. Electrochemical-based tissue-implantable carbon fiber electrodes (CFEs) show great potential in sensing the dynamics of neurochemicals at a sub-second timescale. However, their anti-fouling property, selectivity, and stability pose challenges. Here, we presented a novel strategy to enhance electrode biocompatibility and stability by modifying CFE with a chitosan (CS) film, brain cell membrane (M), and aptamer cholesterol amphiphiles (DNA-cho). We found that CFE was uniformly covered by a cicada-like membrane after being modified. Electrochemical characterizations indicated that DNA-cho-M-CS-CFE exhibited a wide linear range of DA concentration and showed high sensitivity, specificity, and stability. The electrode also presented excellent fouling resistance and biocompatibility. Moreover, the biosensor was used to detect DA in K+-induced brain slices and PC12 cells with a satisfactory stability and sensitivity and to prove that LPS treatment leads to the delayed and decreased release of DA. DNA-cho-M-CS-CFE showed excellent electrochemical performance and unique advantages for long-term in vivo sensing of living cells, thus providing a new feasible scheme for studying neurochemical kinetics and brain diseases.

The electrochemical biosensing strategy for pyocyanin (PYO), a virulent quorum-sensing molecule responsible for Pseudomonas aeruginosa infections, was developed by mimicking its extracellular DNA interaction. Calf thymus DNA (ct-DNA) functionalized amine-containing carbon quantum dots (CQDs) were used as a biomimetic receptor for electrochemical sensing of PYO as low as 37 nM in real urine sample. The ct-DNA-based biosensor enabled the selective measurement of PYO in the presence of other interfering species. Calibration and validation of the PYO sensor platform were demonstrated in buffer solution (0-100 μM), microbial culture media (0-100 μM), artificial urine (0-400 μM), and real urine sample (0-250 μM). The sensor capability was successfully implemented for point-of-care (POC) detection of PYO release from Pseudomonas aeruginosa strains during lag and stationary phases. Cross-reactivity of the sensing platform was also tested in other bacterial species such as Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. Potential clinical implementation of the ct-DNA-based sensor was manifested in detecting the PYO in P. aeruginosa cultured baby diaper and sanitary napkin. Our results highlight that the newly developed ct-DNA-based sensing platform can be used as a potential candidate for real-time POC diagnosis of Pseudomonas aeruginosa infection in clinical samples.

Stress is part of everyone's life and is exacerbated by traumatic events such as pandemics, disasters, violence, lifestyle changes, and health disorders. Chronic stress has many detrimental health effects and can even be life-threatening. Long-term stress monitoring outside of a hospital is often accomplished by measuring heart rate variability. While easy to measure, this digital biomarker has low specificity, greatly limiting its utility. To address this shortcoming, we report a non-invasive, wearable biomolecular sensor to monitor cortisol levels in sweat. Cortisol is a neuroendocrine hormone that regulates homeostasis as part of the stress pathway. Cortisol is detected using an electrochemical sensor functionalized with a pseudoknot-assisted aptamer and a flexible microfluidic sweat sampling system. The skin-worn microfluidic sampler provides rapid sweat collection while separating old and new sweat. The conformation-switching aptamer provides high specificity towards cortisol while being regenerable, allowing it to monitor temporal changes continuously. The aptamer was engineered to add a pseudoknot, restricting it to only two states, thus minimizing the background signal and enabling high sensitivity. An electrochemical pH sensor allows pH-corrected amperometric measurements. Device operation was demonstrated invitro with a broad linear dynamic range (1 pM - 1 μM) covering the physiological range and a sub-picomolar (0.2 pM) limit of detection in sweat. Real-time, on-body measurements were collected from human subjects using an induced stress protocol, demonstrating in-situ signal regeneration and the ability to detect dynamic cortisol fluctuations continuously for up to 90 min. The reported device has the potential to improve prognosis and enable personalized treatments.

The establishment of new mechanisms for target identification and signal amplification continues to drive innovation in electrochemiluminescence (ECL) sensing platforms. In this paper, a novel ECL insulin sensing platform was constructed by utilizing the molecular recognition properties of cucurbit[7]uril. Specifically, the macrocyclic host molecule cucurbit[7]uril was immobilized on the surface of the sensing platform as an identification probe, which could selectively capture insulin according to the inherent properties of the protein N-terminal. Introducing the rigid molecule cucurbit[7]uril into the sensing interface could reduce the influence of the environmental parameters on the sensing system, which provides a reliable guarantee for the accurate detection of insulin. Furthermore, gold nanoclusters were modified by utilizing the molecular recognition properties of cucurbit[7]uril, and used as anode signal probes for ECL sensing platform. The macrocyclic molecules cucurbit[7]uril passivated the surface of the nanoclusters, inhibited the non-radiative relaxation and improved the physical stability of the luminophore, leading to a significant increase in the sensitivity and stability of the ECL probe. The ECL sensing platforms exhibited a linear range from 50.00 fg/mL to 100.0 ng/mL, with a detection limit of 5.44 fg/mL. This study revealed the critical role of cucurbit[7]uril in target recognition and signal amplification, extending the scope of supramolecular applications in ECL.

In this study, a novel split-type electrochemical immunosensor based on controlled release strategy was proposed for sensitive analysis and detection of tumor marker carbohydrate antigen 199 (CA19-9). Specifically, glucose (Glu) was encapsulated in carrier mesoporous silica (MSN) with encapsulation technology, and surface functionalized Zinc sulfide (ZnS) caps were used as "gatekeepers". The complex is formed by encapsulating Glu within MSN with ZnS (ZnS@MSN-Glu) as a signal amplifier labeled on the signal antibody (Ab2). And the Ab2 can detect the presence of antibodies. To reduce the interference of biological analysis, the immune recognition process of ZnS@MSN-Glu-Ab2 bioconjugate and antigen was carried out in 96-well microplate, which did not interfere with the electrochemical analysis process. Therefore, the low sensitivity detection caused by biofouling of nanomaterials and immunoreaction on the testing platform is eliminated. Subsequently, the opening and timed release of mesopores were controlled by external stimuli, the disulfide bond cleavage by dithiothreitol (DTT), and glucose was effectively released. Then nickel cobalt layered double hydroxide (NiCo-LDH) were directly hydrothermally grown on carbon cloth (CC) electrodeposited with copper selenide (CuSe) nanosheets to construct three-dimensional (3D) cactus-like NiCo-LDH/CuSe/CC sensing platform. It can realize the catalytic oxidation of released glucose, triggering glucose-mediated signal amplification. The synergistic effect of the 3D cactus structure and active nanomaterials promotes electron conduction. Taking the detection of carbohydrate antigen CA19-9 as an example, the immunosensor shows a wide linear concentration range (0.001-100 U/mL) with the limit of detection of 0.0005 U/mL, realizing highly sensitive detection of CA19-9. This biosensing technique has considerable advantages and provides an innovative approach for trace detection of other biomarkers.

Improving the electrochemiluminescence (ECL) performance of luminophores is an ongoing research hotspot in the ECL realm. Herein, a high-performance metal-organic framework (MOF)-based ECL material (Ru@Ni3(HITP)2, HITP = 2,3,6,7,10,11-hexaiminotriphenylene) with conductivity- and confinement-enhanced ECL was successfully constructed by using conductive MOF Ni3(HITP)2 as the carrier to graft Ru(bpydc)34- (H2bpydc = 2,2'-bipyridine-4,4'-dicarboxylic acid) into the channels of Ni3(HITP)2. Compared to Ru@Cu3(HITP)2 and Ru@Co3(HITP)2 with relatively low conductivity, the ECL intensity of Ru@Ni3(HITP)2 was prominently increased about 6.76 times and 18.8 times, respectively, which demonstrated that the increase in conductivity induced the ECL enhancement of the MOF-based ECL materials. What's more, the hydrophobic and porous Ni3(HITP)2 can not only effectively enrich the lipophilic tripropylamine (TPrA) coreactants in its channels to enhance the electrochemical oxidation efficiency of TPrA, but also provide a conductive reaction micro-environment to boost the ECL reaction between Ru(bpydc)33- intermediates and TPrA• in confined spaces, thus realizing a remarkable confinement-enhanced ECL. Considering the excellent ECL performance of Ru@Ni3(HITP)2, an ultrasensitive ECL biosensor was prepared based on the Ru@Ni3(HITP)2 ECL indicator combining an exonuclease I-aided target cycling amplification strategy for thrombin determination. The constructed ECL biosensor showcased a wide linear range from 1 fM to 1 nM with a low detection limit of 0.62 fM. Overall, the conductivity- and confinement-enhanced ECL based on Ru@Ni3(HITP)2 provided effective and feasible strategies to enhance ECL performance, which paved a promising avenue for exploring high-efficient MOF-based ECL materials and thus broadened the application scope of conductive MOFs.

Recently, non-metallic SERS-based immunoassay has attracted much attention due to its attractive chemical enhancement (CM), chemical stability, and biocompatibility. Herein, metallic (1T)-semiconductor (2H) mixed-phase magnetic molybdenum disulfide (MoS2) was rationally developed and combined with two-dimensional (2D) graphitic carbon nitride (g-C3N4) nanosheets to realize a SERS-based recyclable immunoassay of CA125. The Fe3O4 core promoted the reliable stacking of MoS2 nanoflakes into a flower-like shape with fully-exposed active surface. Particularly, the existence of 1T phase facilitated a noble-metal-comparable SERS activity due to the high electron density-induced charge transfer process with elevated efficiency. Moreover, a conversion from bulk to 2D nanosheet was swimmingly achieved for g-C3N4 via acid etching, whose large surface area full of active electrons and functional groups triggered an enhancement factor (EF) of 7.8 × 106. Based on a typical sandwich immunostructure, a limit of detection (LOD) as 4.96 × 10-4 IU/mL was demonstrated for CA125 in a recyclable process. Finally, such an immunosensor was employed to analyze clinical samples, indicating its prodigious potentiality in the early recognition and monitoring of cancer.

Activation of Cas13 is exclusively dependent on crRNA-target RNA hybridization according to the canonical mode of Cas13 action. Upon activation Cas13 can cleave both target RNA and any surrounding RNA. The latter has been well adopted by therapeutic gene interference and biosensor development. This work for the first time, rationale designs and validates a multi-component controlled activation system of Cas13 by N-terminus tagging. A composite SUMO tag comprised of His, Twinstrep, and Smt3 tags fully suppresses target dependent activation of Cas13a by interfering with crRNA docking. The suppression releases upon proteases mediated proteolytic cleavage. The modular composition of the composite tag can be altered to fulfill customized response to alternative proteases. The biosensor SUMO-Cas13a is able to resolve a broad concentration range of protease Ulp1 with a calculated LOD of 48.8pg/μL in aqueous buffer. Further, in accordance with this finding Cas13a was successfully programmed to exert target gene knock down preferentially in SUMO protease high cell types. In summary the discovered regulatory component not only fulfills Cas13a based protease detection for the first time, but also delivers a novel strategy for multi-component controlled activation of Cas13a toward temporal and spacial precision.

The increased sensitivity of microelectrode arrays (MEAs) over macroelectrodes for biosensing is well established, and results from reducing the diffusion gradient of the target species to and from the electrode surfaces. The current study describes the fabrication and characterisation of a polymer-based MEA, which exploits the advantages of three dimensionality (3D). Firstly, the unique 3D formfactor promotes release of the gold tips from an inert layer in a controlled fashion, to form a highly reproducible array of microelectrodes in a single step. The 3D topography of the fabricated MEAs significantly enhances the diffusion profile of the target species to the electrode which results in higher sensitivity. Furthermore, the "sharpness" of the 3D structure induces differential current distribution that is focused at the apices of the individual electrodes, reducing the active area, and thereby overcoming the requirement for the electrodes to be sub-micron in size before true MEA behaviour can be achieved. The electrochemical characteristics of the 3D MEAs shows ideal micro-electrode behaviour, with a level of sensitivity of three orders of magnitude greater than that of enzyme-linked immunosorbent assays (ELISA), as the optical based gold standard. The application of the 3D MEAs uses the combination of enzyme-label and substrate approach employed in ELISAs as a generic basis for biosensing and can hence be applied to the plethora of targets that utilise the ELISA approach. As an example, the 3D MEAs are applied to the detection of RNA and demonstrate a level of detection down to single digit picomolar concentrations.

Due to the diversification and complexity of organophosphorus pesticide residues brings great challenges to the detection work. Therefore, we developed a dual-ratiometric electrochemical aptasensor that could detect malathion (MAL) and profenofos (PRO) simultaneously. In this study, metal ions, hairpin-tetrahedral DNA nanostructures (HP-TDN) and nanocomposites were used as signal tracers, sensing framework and signal amplification strategy respectively to develop the aptasensor. Thionine (Thi) labeled HP-TDN (HP-TDNThi) provided specific binding sites for assembling Pb2+ labeled MAL aptamer (Pb2+-APT1) and Cd2+ labeled PRO aptamer (Cd2+-APT2). When the target pesticides were present, Pb2+-APT1 and Cd2+-APT2 were dissociated from the hairpin complementary strand of HP-TDNThi, resulting in reduced oxidation currents of Pb2+ (IPb2+) and Cd2+ (ICd2+), respectively, while the oxidation currents of Thi (IThi) remained unchanged. Thus, IPb2+/IThi and ICd2+/IThi oxidation current ratios were used to quantify MAL and PRO, respectively. In addition, the gold nanoparticles (AuNPs) encapsulated in the zeolitic imidazolate framework (ZIF-8) nanocomposites (Au@ZIF-8) greatly increased the catch of HP-TDN, thereby amplifying the detection signal. The rigid three-dimensional structure of HP-TDN could reduce the steric hindrance effect on the electrode surface, which could greatly improve the recognition efficiency of the aptasensor for the pesticide. Under the optimal conditions, the detection limits of the HP-TDN aptasensor for MAL and PRO were 4.3 pg mL-1 and 13.3 pg mL-1, respectively. Our work proposed a new approach to fabricating a high-performance aptasensor for simultaneous detection of multiple organophosphorus pesticides, opening a new avenue for the development of simultaneous detection sensors in the field of food safety and environmental monitoring.

Multiple studies showed that metabolic disorders play a critical role in respiratory infectious diseases, including COVID-19. Metabolites contained in small extracellular vesicles (sEVs) are different from those in plasma at the acute stage, while the metabolic features of plasma sEVs of COVID-19 survivors remain unknown. Here, we used a nanopore membrane-based microfluidic chip for plasma sEVs separation, termed ExoSEC, and compared the sEVs obtained by UC, REG, and ExoSEC in terms the time, cost, purity, and metabolic features. The results indicated the ExoSEC was much less costly, provided higher purity by particles/proteins ratio, and achieved 205-fold and 2-fold higher sEVs yield, than UC and REG, respectively. Moreover, more metabolites were identified and several signaling pathways were significantly enriched in ExoSEC-sEVs compared to UC-sEVs and REG-sEVs. Furthermore, we detected 306 metabolites in plasma sEVs using ExoSEC from recovered asymptomatic (RA), moderate (RM), and severe/critical COVID-19 (RS) patients without underlying diseases 3 months after discharge. Our study demonstrated that COVID-19 survivors, especially RS, experienced significant metabolic alteration and the dysregulated pathways mainly involved fatty acid biosynthesis, phenylalanine metabolism, etc. Metabolites of the fatty acid biosynthesis pathway bore a significantly negative association with red blood cell counts and hemoglobin, which might be ascribed to hypoxia or respiratory failure in RM and RS but not in RA at the acute stage. Our study confirmed that ExoSEC could provide a practical and economical alternative for high throughput sEVs metabolomic study.

The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.

Magnetic quantum dot (QD) barcode holds great potential for automatic suspension array and rapid point-of-care detection since it enables simultaneous target encoding, enrichment and separation. However, a serious obstacle to enhancing the encoding capacity of magnetic QD microbeads (MBs) is the fluorescence quenching of magnetic nanoparticles (MNPs) to quantum dots (QDs) in the visible wavelength range due to the broad and strong optical absorption spectrum of MNPs. Here, we report Fe3O4/TiO2 core/shell MNPs and CdSe/ZnS QDs for the construction of dual-function magnetic QD barcodes. Fe3O4/TiO2 MNPs can significantly inhibit fluorescence quenching because the weak absorption of visible light by the TiO2. The two-dimension barcode library of 30 magnetic QD barcodes was constructed based on Fe3O4/TiO2 MNPs and CdSe/ZnS QDs. Moreover, the magnetic QD barcodes showed high sensitivity for the multiplex detection of four tumor markers, cancer antigen 125 (CA125), cancer antigen 199 (CA199), alpha-fetoprotein (AFP), and neuron specific enolase (NSE) with detection limits of 0.89 KU/L, 0.72 KU/L, 0.05 ng/mL, and 0.15 ng/mL, respectively. This bifunctional magnetic QD barcodes are promising for automatic high-sensitivity multiplex bioassay.

Herein, we implemented RNA-cleaving DNAzymes specific for the endogenous protein of breast cancer cells (MDA-MB -231) and programmed for electrochemical detection. Thionine-modified gold nanoparticles and modified magnetic nanoparticles are attached to the two ends of the DNAzyme molecule. The prepared probe is pulled to the surface of the electrode with the help of a magnetic field, and the signal caused by the electrochemical activity of thionine is observed on the surface of the electrode. The presence of a covalent gold nanoparticle-thionine hybrid as a highly electroactive/enhanced electrochemical label ensures a strong detection signal. After addition of the enzyme activator cofactor (MDA-MB -231 cytoplasmic cell protein), it reacts with the catalytic core of the enzyme sequence in the DNAzyme molecule and triggers the cleavage reaction in the substrate sequence of the DNAzyme molecule. During this process, the gold nanoparticle-thionine labels are detached from the probe and released into the solution. Inductive removal of gold nanoparticles leads to a decrease in the current related to the reduction of thionine on the electrode surface. The results show that this biosensor can detect this protein marker in the linear range of (1.0E-06 to 1.0E+01) pg/ml, with a detection limit (1.0129E-07 pg/ml), using differential pulse voltammetry as a measuring technique. As well as, electrochemical impedance spectroscopy (EIS).

Simultaneous detection of multiple biomarkers is always an obstacle in immunohistochemical (IHC) analysis. Herein, a straightforward spectroscopy-driven histopathologic approach has emerged as a paradigm of Raman-label (RL) nanoparticle probes for multiplex recognition of pertinent biomarkers in heterogeneous breast cancer. The nanoprobes are constructed by sequential incorporation of signature RL and target specific antibodies on gold nanoparticles, which are coined as Raman-Label surface enhanced Raman scattering (RL-SERS)-nanotags to evaluate simultaneous recognition of clinically relevant breast cancer biomarkers i.e., estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor2 (HER2). As a foot-step assessment, breast cancer cell lines having varied expression levels of the triple biomarkers are investigated. Subsequently, the optimized detection strategy using RL-SERS-nanotags is subjected to clinically confirmed, retrospective formalin-fixed paraffin embedded (FFPE) breast cancer tissue samples to fish out the quick response of singleplex, duplex as well as triplex biomarkers in a single tissue specimen by adopting a ratiometric signature RL-SERS analysis which enabled to minimize the false negative and positive results. Significantly, sensitivity and specificity of 95% and 92% for singleplex, 88% and 85% for duplex, and 75% and 67% for triplex biomarker has been achieved by assessing specific Raman fingerprints of the respective SERS-tags. Furthermore, a semi-quantitative evaluation of HER2 grading between 4+/2+/1+ tissue samples was also achieved by the Raman intensity profiling of the SERS-tag, which is fully in agreement with the expensive fluorescent in situ hybridization analysis. Additionally, the practical diagnostic applicability of RL-SERS-tags has been achieved by large area SERS imaging of areas covering 0.5-5 mm2 within 45 min. These findings unveil an accurate, inexpensive and multiplex diagnostic modality envisaging large-scale multi-centric clinical validation.

Seasonal outbreaks of respiratory viral infections remain a global concern, with increasing morbidity and mortality rates recorded annually. Timely and false responses contribute to the widespread of respiratory pathogenic diseases owing to similar symptoms at an early stage and subclinical infection. The prevention of emerging novel viruses and variants is also a big challenge. Reliable point-of-care diagnostic assays for early infection diagnosis play a critical role in the response to threats of epidemics or pandemics. We developed a facile method for specifically identifying different viruses based on surface-enhanced Raman spectroscopy (SERS) with pathogen-mediated composite materials on Au nanodimple electrodes and machine learning (ML) analyses. Virus particles were trapped in three-dimensional plasmonic concave spaces of the electrode via electrokinetic preconcentration, and Au films were simultaneously electrodeposited, leading to the acquisition of intense and in-situ SERS signals from the Au-virus composites for ultrasensitive SERS detection. The method was useful for rapid detection analysis (<15 min), and the ML analysis for specific identification of eight virus species, including human influenza A viruses (i.e., H1N1 and H3N2 strains), human rhinovirus, and human coronavirus, was conducted. The highly accurate classification was achieved using the principal component analysis-support vector machine (98.9%) and convolutional neural network (93.5%) models. This ML-associated SERS technique demonstrated high feasibility for direct multiplex detection of different virus species for on-site applications.

Numerous bacteria can cause water- and foodborne diseases and are often found in bacterial mixtures, making their detection challenging. Specific bioreceptors or selective growth media are necessary for most bacterial detection methods. In this work, we collectively used five quorum sensing-based peptides identified from bacterial biofilms to identify 10 different bacterial species (Bacillus subtilis, Campylobacter jejuni, Enterococcus faecium, Escherichia coli, Legionella pneumophila, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium, Staphylococcus aureus, Vibrio parahaemolyticus) and their mixtures in water and milk. Four different machine learning classification methods were used: k-nearest neighbors (k-NN), decision tree (DT), support vector machine (SVM), and eXtreme Gradient Boosting (XGBoost). Peptides were crosslinked to submicron particles, and peptide-bacteria interactions on paper microfluidic chips caused the particle aggregation. A wireless, pocket fluorescence microscope (interfaced with a smartphone) counted such particle aggregations. XGBoost showed the best accuracy of 83.75% in identifying bacterial species from water samples using 320 different datasets and 91.67% from milk samples using 140 different datasets (5 peptide features per dataset). Each peptide's contribution to correct classification was evaluated. The results were concentration-dependent, allowing the identification of a dominant species from bacterial mixtures. Using XGBoost and the previous milk database, we tested 14 blind samples of various bacterial mixtures in milk samples, with an accuracy of 81.55% to predict the dominant species. The entire process could be completed within a half hour. The demonstrated system can provide a handheld, low-cost, easy-to-operate tool for potential hygiene spot-checks, public health, or personal healthcare.

A highly invasive subpopulation of circulating tumor cells (CTCs) may constitute seeds for metastases, which are therefore considered functional CTCs. However, there are few effective strategies to detect CTCs based on invasive phenotypes. Herein, we focused on functional CTCs with high invasiveness and designed an integrated microfluidic system to differentiate the invasive potential of CTCs for more accurate metastasis prediction. By combining size-based enrichment and invasiveness-based analysis, the system managed to continuously remove most hemocytes by 8 μm gaps and analyze the invasiveness of the enriched CTCs by Matrigel loading. In addition to a device, a single pump and a Petri dish were included to provide an FBS gradient for driving cell invasion and maintain a long-term cell culture. The system successfully identified functional CTCs derived from different types of cancer patients, including colorectal, kidney and bladder cancer patients, using whole blood without any sample pretreatment process. Within 28 cases of colorectal cancer patients, functional CTCs were detected in 61.54% of patients with metastases, along with stronger invasiveness evaluated by migration/invasion distance than those from patients without metastases (P < 0.05). Furthermore, one bladder cancer patient was diagnosed with recurrence six months after detection, indicating the excellent value for cancer metastases prediction. In addition, great phenotypic heterogeneity of CTCs was also observed at the single-cell level, including invasion, proliferation and dormancy, which provided an effective strategy for metastasis prediction based on CTC function as a single cell.