Using microfluidic paper-based analytical devices has attracted considerable attention in recent years. This is mainly due to their low cost, availability, portability, simple design, high selectivity, and sensitivity. Owing to their specific substrates and catalytic functions, enzymes are the most commonly used bioactive agents in μPADs. Enzymatic μPADs are various in design, fabrication, and detection methods. This paper provides a comprehensive review of the development of enzymatic μPADs by considering the methods of detection and fabrication. Particularly, techniques for mass production of these enzymatic μPADs for use in different fields such as medicine, environment, agriculture, and food industries are critically discussed. This paper aims to provide a critical review of μPADs and discuss different fabrication methods as the central parts of the μPADs production categorized into printable and non-printable methods. In addition, state-of-the-art technologies such as fully printed enzymatic μPADs for rapid, low-cost, and mass production and improvement have been considered.

Space exploration is facing a new era in view of the planned missions to the Moon and Mars. The development and the in-flight validation of new technologies, including analytical and diagnostic platforms, is pivotal for exploring and inhabiting these extreme environments. In this context, biosensors and lab-on-chip devices can play an important role in many situations, such as the analysis of biological samples for assessing the impact of deep space conditions on man and other biological systems, environmental and food safety monitoring, and the search of molecular indicators of past or present life in extra-terrestrial environments. Small satellites such as CubeSats are nowadays increasingly exploited as fast and low-cost platforms for conducting in-flight technology validation. Herein, we report the development of a fully autonomous lab-on-chip platform for performing chemiluminescence-based bioassays in space. The device was designed to be hosted onboard the AstroBio CubeSat nanosatellite, with the aim of conducting its in-flight validation and evaluating the stability of (bio)molecules required for bioassays in a challenging radiation environment. An origami-like microfluidic paper-based analytical format allowed preloading all the reagents in the dried form on the paper substrate, thus simplifying device design and analytical protocols, facilitating autonomous assay execution, and enhancing the stability of reagents. The chosen approach should constitute the first step to implement a mature technology with the aim to conduct life science research in space (e.g., for evaluation the effect of deep space conditions on living organisms or searching molecular evidence of life) more easily and at lower cost than previously possible.

Wearable biosensors (WB) are currently attracting considerable interest for rapid detection and monitoring of biomarkers including metabolites, protein, and pathogen in bodily fluids (e.g., sweat, saliva, tears, and interstitial fluid). Another branch of WB termed wearable nucleic acid testing (NAT) is blossoming thanks to the development of microfluidic technology and isothermal nucleic acid amplification technique (iNAAT); however, there are only few reports on this. The wearable NAT is an emerging field of point-of-care (POC) diagnostics, and holds the promise for time-saving self-diagnosis, and evidence-based surveillance of infectious diseases in remote or low-resource settings. The use of wearable NAT can also be advanced to include molecular diagnosis, the identification of cancer biomarkers, genetic abnormalities, and other aspects. The wearable NAT provides the potential for evidence-based surveillance of infectious diseases when combined with internet connectivity and App software. To make the wearable NAT accessible to the end users, however, improvements must be made to the fabrication, cost, speed, sensitivity, specificity, sampling, iNAAT, analyzer, and a few other features. So, in this paper, we looked at the wearable NAT's most recent development, identified its difficulties, and defined its potential for managing infectious diseases quickly in the future. This is the wearable NAT review's first effort. We expect that this article will provide the concise resources needed to develop and deploy an efficient wearable NAT system.

Cholera is a highly contagious and lethal waterborne disease induced by an infection with Vibrio cholerae (V. cholerae) secreting cholera toxin (CTx). Cholera toxin subunit B (CTxB) from the CTx specifically binds with monosialo-tetra-hexosyl-ganglioside (GM1) found on the exterior cell membrane of an enterocyte. Bioinspired by the pathological process of CTx, we developed an electrochemical biosensor with GM1-expressing Caco-2 cell membrane (CCM) on the electrode surface. Briefly, the electrode surface was functionalized with CCM using the vesicle fusion method. We determined the CTxB detection performances of Caco-2 cell membrane-coated biosensor (CCB) using electrochemical impedance spectroscopy (EIS). the CCB had an excellent limit of detection of ∼11.46 nM and a detection range spanning 100 ng/mL - 1 mg/mL. In addition, the CCB showed high selectivity against various interfering molecules, including abundant constituents of intestinal fluid and various bacterial toxins. The long-term stability of the CCBs was also verified for 3 weeks using EIS. Overall, the CCB has excellent potential for practical use such as point-of-care and cost-effective testing for CTxB detection in developing countries.

This work examines the suitability of graphene-based 3D-printed nanocomposite bioelectronics as innovative systems to in situ monitor and evaluate both breast cancer cell adhesion and the chemosensitivity of anti-cancer drugs. With this aim, 3D-printed nanocomposite graphene electrodes (3D-nGEs) -made of a commercially available graphene/polylactic acid filament- have been covalently biofunctionalized with an extracellular matrix protein (i.e., fibronectin) by exploiting the carbon reactivity of 3D-nGEs. The specificity and selectivity of the developed electrochemical system to monitor breast cancer cell adhesion has been tested via electrochemical impedance spectroscopy (EIS). Importantly, the resulting 3D-printed bioelectronic system displayed excellent accuracy for the rapid screening of anti-cancer drugs, which exactly corresponded with the results achieved by the standard optical method, while having the advantage of employing a label-free approach. In light of the current state-of-the-art in the field, this proof-of-concept connects electronics to biological systems within 3D printing technology, providing the bases for the sustainable and cost-effective manufacturing of graphene-based 3D-printed nanocomposite bioelectronics to simulate in vivo microenvironments using in situ and real time electronic output signals.

Ketamine is an organic drug with weak electrochemical activity, which makes it difficult to directly detect by electrochemical methods. Herein, an electrochemical sensor, with excellent detection sensitivity, is proposed for direct detection of ketamine based on a weakly conductive poly-L-cysteine molecularly imprinted membrane. Poly-L-cysteine molecularly imprinted membrane sensor (poly-L-Cys-KT-MIM/GCE) is obtained using L-cysteine as a functional monomer and ketamine as a template molecule based on electropolymerization. The green and highly active cysteine is selected as a functional monomer during electropolymerization, which cannot only achieve specific recognition but also improve detection sensitivity. Furthermore, the oxidation mechanism and fingerprint of ketamine on the electrode surface are established by analyzing the corresponding oxidation products using high/resolution mass spectrometry, which will help to promote the application of electrochemistry in the rapid detection of drugs. Under optimal conditions, the as-designed sensor demonstrated a linear response to ketamine within the range of 5.0 × 10-7 to 2.0 × 10-5 mol L-1 and a detection limit of 1.6 × 10-7 mol L-1. The proposed method exhibited excellent performance from the viewpoints of selectivity, sensitivity and stability. Notably, the sensor rendered excellent reliability and could be used for the detection of target analytes in hair and urine samples with high recovery rates.

The key factor that causes glioma invasion and metastasis is circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT). Effective analysis of EMT-CTCs can provide an important foundation for early detection and prognosis monitoring of glioma, but the changes in the biomarkers of CTCs in different states of EMT make detection difficult. In this study, we developed a multivalent aptamer nanoscaffold-based electrochemical cytosensor (MAS-cytosensor) to efficiently detect EMT-CTCs. The two chains forming the MAS are composed of a specific aptamer detector, a binding region for DNA self-assembly, and a foothold for interface anchoring. When target CTCs exist, the bisaptamer detector on MAS can sensitively identify CTCs and pull them to the electrode surface, generating electrochemical signals. It has been demonstrated that the MAS-cytosensor can not only detect EMT-CTCs sensitively (detection limit of 6 cells/mL in buffer), but also allows for further downstream analysis after release with high viability. Overall, this cytosensor provides a reliable detection solution for CTCs regardless of their EMT status, and provides an efficient method for in-depth study role of the post-EMT CTCs in clinical application and metastasis mechanisms.

Foreign body response (FBR), inflammation, and fibrotic encapsulation of neural implants remain major problems affecting the impedance of the electrode-tissue interface and altering the device performance. Adhesion of proteins and cells (e.g., pro-inflammatory macrophages, and fibroblasts) triggers the FBR cascade and can be diminished by applying antifouling coatings onto the implanted devices. In this paper, we report the deposition and characterization of a thin (±6 nm) sulfobetaine-based coating onto microfabricated platinum electrodes and cochlear implant (CI) electrode arrays. We found that this coating has stable cell and protein-repellent properties, for at least 31 days in vitro, not affected by electrical stimulation protocols. Additionally, its effect on the electrochemical properties relevant to stimulation (i.e., impedance, charge injection capacity) was negligible. When applied to clinical CI electrode arrays, the film was successful at inhibiting fibroblast adhesion on both the silicone packaging and the platinum/iridium electrodes. In vitro, in fibroblast cultures, coated CI electrode arrays maintained impedance values up to five times lower compared to non-coated devices. Our studies demonstrate that such thin sulfobetaine containing layers are stable and prevent protein and cell adhesion in vitro and are compatible for use on CI electrode arrays. Future in vivo studies should be conducted to investigate its ability to mitigate biofouling, fibrosis, and the resulting impedance changes upon long-term implantation in vivo.

Herein, photosystem II (PSII), extracted from spinach, is used for the first time as an efficient and green sensitizer for a photobioanode in a photoelectrochemical glucose biofuel cell (PBFC) setup. The concept is based on the formation of hemin-catalyzed luminol chemiluminescence (CL) after the enzymatic oxidation of glucose and the simultaneous production of hydrogen peroxide by glucose oxidase. The photosynthetic enzyme PSII, combined with an osmium polymer serving as mediator and photosensitizer, is immobilized and wired on microporous carbonaceous material (MC) for the chemiluminescence-induced oxidation of water to O2 at the photobioanode (GCE|MC|Os polymer|PSII). Also, bilirubin oxidase immobilized on multiwalled carbon nanotubes (MWCNTs) coated electrode (GCE|MWCNT|BOx) serves as a biocathode. The photoelectrochemical biofuel cell (PBFC) is applied to a biosensor model system to validate the appropriateness of such a bioanode operating in a self-powered mode. Os redox polymer attached to MCs provides abundant PSII immobilization and a reliable electron transfer pathway. The well-matching energy levels of photosensitive entities reduce recombination phenomena while MC enhances the charge collection. Substantial photocatalytic water oxidation was observed under CL due to the well-matched CL emission and PSII absorption. The electrode is rationally designed to gain the maximum luminol CL power for the photobioanode. The open circuit potential of PBFC linearly increased with the CL power intensity and, in turn, glucose concentrations in the range of 0-6 mmol L-1. The PBFC yielded an OCP of 0.531 V in 30 mmolL-1 glucose. The study may open a new horizon to the green and pioneering PEC biosensing realm.

Nanowires have substantial potential as the sensor component in electronic sensing devices. However, surface functionalization of traditional nanowire and nanotube materials with short peptides that increase sensor selectivity and sensitivity requires complex chemistries with toxic reagents. In contrast, microorganisms can assemble pilin monomers into protein nanowires with intrinsic conductivity from renewable feedstocks, yielding an electronic material that is robust and stable in applications, but also biodegradable. Here we report that the sensitivity and selectivity of protein nanowire-based sensors can be modified with a simple plug and play genetic approach in which a short peptide sequence, designed to bind the analyte of interest, is incorporated into the pilin protein that is microbially assembled into nanowires. We employed a scalable Escherichia coli chassis to fabricate protein nanowires that displayed either a peptide previously demonstrated to effectively bind ammonia, or a peptide known to bind acetic acid. Sensors comprised of thin films of the nanowires amended with the ammonia-specific peptide had a ca. 100-fold greater response to ammonia than sensors made with unmodified protein nanowires. Protein nanowires with the peptide that binds acetic acid yielded a 4-fold higher response than nanowires without the peptide. The protein nanowire-based sensors had greater responses than previously reported sensors fabricated with other nanomaterials. The results demonstrate that protein nanowires with enhanced sensor response for analytes of interest can be fabricated with a flexible genetic strategy that sustainably eliminates the energy, environmental, and health concerns associated with other common nanomaterials.

In this work, a stimuli-responsive SERS biosensor was fabricated for tetracycline (TTC) by "signal-on" strategy using (EDTA)-driven polyethyleneimine grafted calcium carbonate (PEI@CaCO3) microcapsule and chitosan-Fe magnetic microbeads (CS@FeMMs). Initially, aptamer conjugated magnetic-bead CS@FeMMs@Apt with superparamagnetism and excellent biocompatibility was employed as capture probe, which facilitated the rapid and easy magnetic separation. Subsequently, the PEI cross-linked layer and aptamer network layer were constructed onto the outer layer of CaCO3@4-ATP microcapsule to form sensing probes (PEI@CaCO3@4-ATP@Apt) via the layer-by-layer assembly method. In the presence of TTC, a sandwich SERS-assay was exploited by aptamer recognition induced target-bridged strategy. When the solution of EDTA was added, the core layer of CaCO3 would be dissolved quickly, destroying the microcapsule to release 4-ATP. The released 4-ATP could be quantitatively monitored by dripping the supernatant onto the AuNTs@PDMS SERS platform, resulting in a strong Raman "signal-on". Under the optimal conditions, a good linear relationship was established with a correlation coefficient (R2) of 0.9938 and a LOD of 0.03 ng/mL. Additionally, the application capacity of the biosensor to detect TTC was also affirmed in food matrixes, and the results were consistent with the standard ELISA method (P > 0.05). Hence, this SERS biosensor affords extensive application prospects for TTC detection with multiple merits such as high sensitivity, environment friendliness, and high stability.

The separation of the superimposed electrochemical signals of intracellular guanine (G) and xanthine (X) is difficult, which is great obstacle to the application of cell electrochemistry. In this paper, independent functional modules, G-functional module (G-FM) and X-functional module (X-FM), were constructed by molecular imprinting technology for sensitive detection of G and X without mutual interference, then integrated in dual-functional module cellular electrochemical sensing platform (DMCEP) as signal sensing units. DMCEP transmitted signals of G and X in cells synchronously to two windows by two signal sensing channels, and achieved the separation of superimposed signals of G and X in cells. DMCEP exhibited satisfactory reproducibility with relative standard deviation (RSD) of 3.10 and 2.22 %, repeatability with RSD of 3.72 and 3.05 % for G and X detection, and detection limit 0.05 μΜ for G and 0.06 μΜ for X. Good linear relationships between cell concentrations and the signals of G and X on DMCEP were shown in range of 0.75-85 × 106 and 3-85 × 106 cells/mL, respectively. The growth of MCF-7 cells was tracked by DMCEP, and showed consistent trend with the cell counting method, while the change of cell viability from lag to logarithmic phase captured by DMCEP was earlier than that of cell counting method. This strategy provided the foundation for the establishment of the cell viability electrochemical detection method, and new insights into the simultaneous recording of other analyses with superimposed peak positions and the simultaneous tracking of multiple biomarkers.

Monoclonal antibody (mAb) therapy is one of the most promising immunotherapies that have shown the potential to prevent or neutralize the effects of COVID-19 in patients at very early stages, with a few formulations recently approved by the European and American medicine agencies. However, a main bottleneck for their general implementation resides in the time-consuming, laborious, and highly-specialized techniques employed for the manufacturing and assessing of these therapies, excessively increasing their prices and delaying their administration to the patients. We propose a biomimetic nanoplasmonic biosensor as a novel analytical technique for the screening and evaluation of COVID-19 mAb therapies in a simpler, faster, and reliable manner. By creating an artificial cell membrane on the plasmonic sensor surface, our label-free sensing approach enables real-time monitoring of virus-cell interactions as well as direct analysis of antibody blocking effects in only 15 min assay time. We have achieved detection limits in the 102 TCID50/mL range for the study of SARS-CoV-2 viruses, which allows to perform neutralization assays by only employing a low-volume sample with common viral loads. We have demonstrated the accuracy of the biosensor for the evaluation of two different neutralizing antibodies targeting both Delta and Omicron variants of SARS-CoV-2, with half maximal inhibitory concentrations (IC50) determined in the ng/mL range. Our user-friendly and reliable technology could be employed in biomedical and pharmaceutical laboratories to accelerate, cheapen, and simplify the development of effective immunotherapies for COVID-19 and other serious infectious diseases or cancer.

Current biosensing detection assays were developed to focus on rapid, low-cost, stable detection for clinical diagnosis and food safety monitoring. In this work, a novel portable cartridge voltage-sensitive micropump immunosensor (CVMS) biosensing device based on the integration of the microchannel circuit biosensing principle and polydopamine (PDA) was presented for rapid and sensitive detection of pathogenic factors in real samples at trace levels. The CVMS can sensitively evaluate voltage signal changes caused by clogging effects in the closed-loop circuit when the insulated microspheres pass through the microchannel. The targets could trigger the immune reaction between antibody-antigens that leads to the change in the concentration of horseradish peroxidase (HRP). And the HRP was further employed to catalyze the polymerization of dopamine into PDA, resulting in the rapid formation of the magnetic @PDA nanoparticles (MNP@PDA) with core-shell structures. The abundant functional groups on the MNP@PDA surface can efficiently adsorb polystyrene microspheres, thus causing changes in the number of polystyrene microspheres (PS). The CVMS can accurately monitor the change of PS with a portable device, which weighs less than 0.8 kg and costs only $50. The completion of CVMS takes 90 min to complete. The limit of detection of this immunosensor for procalcitonin and ochratoxin A were 42 pg/mL and 77 pg/mL, respectively, which improved about 15 folds and 38 folds, respectively, than those of enzyme-linked immunosorbent assay.

In recent years, paper-based nucleic acid sensors have been demonstrated for the ability to detect DNA and RNA molecules extracted from viruses and bacteria. In clinical samples, these nucleic acids are mostly encapsulated in lipid membranes and need to be released before being analyzed using paper-based sensors. For the nucleic acid amplification tests (NAATs), it is also desirable to remove the interfering molecules that can inhibit the nucleic acid amplification. To achieve a field deployable NAAT, we report a portable sensor system that combines the thermolysis and paper-based NAATs to detect target RNA molecules carried by viral and exosomal nanoparticles. The sensor cartridge includes a lysis chamber with a pressure-controlled diaphragm valve, paper flow channels, and three paper-based NAAT reaction chambers to extract, transport, and detect nucleic acids respectively. A compact instrument was prototyped to automate the assay, collect fluorescence images of the nucleic acid amplification, and generate amplification curves for NAATs. The pump-free and paper-based sensor achieved quantitative analysis of influenza A virus (IAV) RNA and exosome microRNA within 1 h, with the lowest detect concentration of 104 TCID50/mL and 106 EV/mL for IAV and exosome, respectively. Owing to the advantages of easy storage, simple operation, and low cost, such as system has great potential to be used as a point-of-care test for in-field diagnosis of viral and bacterial infections.

Herein, a novel near-infrared (NIR) light-driven photoelectrochemical (PEC) biosensor based on NaYF4:Yb3+, Er3+@Bi2MoO6@Bi (NYF@BMO@Bi) nanocomposites was elaborately developed to achieve highly sensitive detection of microRNA-155 (miRNA-155). To realize signal enhancement, the coupled plasmonic bismuth (Bi) nanoparticles were constructed as an energy relay to facilitate the transfer of energy from NaYF4:Yb3+, Er3+ to Bi2MoO6, ultimately enabling the efficient separation of electron-hole pairs of Bi2MoO6 under the irradiation of a 980 nm laser. For constructing biosensing system, the initial signal was firstly amplified after the addition of alkaline phosphatase (ALP) in conjunction with the biofunctionalized NYF@BMO@Bi nanocomposites, which could catalyze the conversion of ascorbic acid 2-phosphate into ascorbic acid, and then consumed the photoacoustic holes created on the surface of Bi2MoO6 for the enlarging photocurrent production. Upon addition of target miRNA-155, the cascade signal amplification process was triggered while the ALP-modified DNA sequence was replaced and then followed by the initiation of a simulated biocatalytic precipitation reaction to attenuate the photocurrent response. On account of the NIR-light-driven and cascade amplifications strategy, the as-constructed biosensor was successfully utilized for the accurate determination of miRNA-155 ranging from 1 fM to 0.1 μM with a detection limit of 0.32 fM. We believed that the proposed nanocomposites-based NIR-triggered PEC biosensor could provide a promising platform for effective monitoring other tumor biomarkers in clinical diagnostics.

Point of care testing (POCT) has important clinical significance for the diagnosis and prognosis evaluation of diseases. At present, the biosensor based on CRISPR/Cas12a has become a powerful diagnostic tool due to its high sensitivity. However, CRISPR/Cas12a requires PAM sequence to recognize target double strand and only can recognize specific sequence, so it is not universal. The current RNA detection techniques either lack consideration for specificity and universality, are expensive and difficult, or both. Therefore, it is crucial to create a CRISPR/Cas12a-based RNA detection system that is easy to use, cheap, specific, and universal in order to further its use in molecular diagnostics. Here, we established a DNA circuit-mediated PAM-independent CRISPR/Cas12a coupled PolyA-rolling circle amplification for RNA detection biosensor, namely DCPRBiosensor. The DCPRBiosensor not only functions as a simple, inexpensive, and highly sensitive RNA detection sensor, but it also boasts innovative specificity and universality features. More importantly, DCPRBiosensor removes the PAM restriction of CRISPR/Cas12a. The DCPRBiosensor's detection limit reached 100 aM and it had a linear relationship between 100 aM and 10 pM. We detected four piRNAs to verify the universality and stability of DCPRBiosensor. Then, we verified that DCPRBiosensor has good discrimination ability for single-base mismatch. Finally, we successfully detected piRNA in DLD-1 and HCT-116 cells and urine mixed samples within 4.5 h. In conclusion, we believe that DCPRBiosensor will have a substantial impact on both the development of CRISPR/as12a's applications and the investigation of the clinical value of piRNA.

A highly sensitive, circulating tumor cell (CTC)-based liquid biopsy was used to monitor gastrointestinal cancer patients during treatment to determine if CTC abundance was predictive of disease recurrence. The approach used a combination of biomimetic cell rolling on recombinant E-selectin and dendrimer-mediated multivalent immunocapture at the nanoscale to purify CTCs from peripheral blood mononuclear cells. Due to the exceptionally high numbers of CTCs captured, a machine learning algorithm approach was developed to efficiently and reliably quantify abundance of immunocytochemically-labeled cells. A convolutional neural network and logistic regression model achieved 82.9% true-positive identification of CTCs with a false positive rate below 0.1% on a validation set. The approach was then used to quantify CTC abundance in peripheral blood samples from 27 subjects before, during, and following treatments. Samples drawn from the patients either prior to receiving radiotherapy or early in chemotherapy had a median 50 CTC ml-1 whole blood (range 0.6-541.6). We found that the CTC counts drawn 3 months post treatment were predictive of disease progression (p = .045). This approach to quantifying CTC abundance may be a clinically impactful in the timely determination of gastrointestinal cancer progression or response to treatment.

By introducing the computer numerical control (CNC) engraving technology, this study fabricated the reusable CNC-fabricated membrane-less laminar flow microfluidic MFC (LMMFC) to develop the bioelectrochemical sensor and power source simultaneously. To verify its applicability, optimization of electroactive bacteria (EAB) cultivation and laminar-flow formation, performance of power density and long-term operation, and detection of Cr(VI) were evaluated. Results of EAB optimization showed under lower external resistance, shorter start-up time of current production, larger oxidation current, denser microbial distribution, and a higher percentage of Geobacter spp. were observed. Results of the laminar-flow operation indicated that increasing the density difference between two solutions and raising the anode flow velocity can minimize the interference of the diffusion zone. The power output of LMMFC could reach 2085 mW m-2 and achieve long-term stability for current production (∼150 h). Regarding the detection of Cr(VI), low-concentration (0.1∼1 ppm) and high-concentration (1-10 ppm) ranges reached the linear coefficient of determination of 0.98 and 0.97, respectively. Overall, these results suggest that an LMMFC which can both act as the power source and biosensor was successfully developed, showing potential for future self-power application.

To date, it is still a challenge for high-performance photoelectrochemical (PEC) assay of low-abundance adenosine deaminase (ADA) in fundamental research and clinical diagnosis. Herein, phosphate-functionalized Pt/TiO2 (termed PO43-/Pt/TiO2) was prepared as ideal photoactive material to develop a split-typed PEC aptasensor for detection of ADA activity, coupled by a Ru(bpy)32+ sensitization strategy. We critically studied the effects of the PO43- and Ru(bpy)32+ on the detection signals, and discussed the signal-amplified mechanism. Specifically, hairpin-structured adenosine (AD) aptamer was splited into single chain via ADA-induced catalytic reaction, and subsequently hybridized with complementary DNA (cDNA, initially coating on magnetic beads). The in-situ formed double-stranded DNA (dsDNA) was further intercalated by more Ru(bpy)32+ to amplify the photocurrents. The resultant PEC biosensor showed a broader linear range of 0.05-100 U L-1 and a lower limit of detection (0.019 U L-1), which can fill the blank for analysis of ADA activity. This research would provide some valuable insights for building advanced PEC aptasensors in ADA-related research and clinical diagnosis.

DNA nanomachines have shown potential application in the construction of various biosensors. Here, an electrochemiluminescence biosensor for the sensitive detection of miRNA-21 were reported based on three-dimensional (3D) DNA nanomachine and duplex-specific nuclease (DSN)-mediated target recycle amplification strategy. First, the bipedal DNA walkers were obtained by DSN-mediated digestion reaction initiated by target miRNA-21.3D DNA tracks were prepared by modifying Fe3O4 magnetic beads (MBs) with ferrocene-labeled DNA (Fc-DNA). The produced DNA walkers autonomously moved along 3D DNA tracks powered by nicking endonuclease. During the movement, ferrocene-labeled DNA was cleaved, resulting in large amounts of Fc-labeled DNA fragments away from the MBs surface. Finally, the liberated Fc-labeled DNA fragments were dropped on the C-g-C3N4 modified electrode surface, leading to the quenching of C-g-C3N4 electrochemiluminescence (ECL). Benefiting from the dual amplification strategy of 3D DNA nanomachine and DSN-mediated target recycling, the developed ECL biosensor exhibited an excellent performance for miRNA-21 detection with a wide linear range of 10 fM to 10 nM and a low detection limit of 1.0 fM. This work offers a new thought for the application of DNA walkers in the construction of various biosensors.

For the diagnosis and therapy of small cell lung cancer (SCLC), the accurate and sensitive determination of neuron-specific enolase (NSE) content is crucial. This work outlines a dual-quenching electrochemiluminescence resonance energy transfer (ECL-RET) immunosensor based on the double quenching effects of iron base metal organic frameworks (FeMOFs) loaded with small sized CuO nanoparticles (FeMOFs-sCuO) towards CoPd nanoparticles (CoPdNPs) enhanced porous g-C3N4 (P-C3N4-CoPdNPs). To be specific, we prepared a porous g-C3N4 (P-C3N4) which has a rich porous structure, and significantly increased the specific surface area and the number of reaction sites of P-C3N4. Meanwhile, the CoPdNPs were loaded onto P-C3N4 to improve the ECL luminescence property of P-C3N4/K2S2O8 system through acting as a coreaction accelerator. In addition, the ultraviolet-visible (UV-vis) absorption spectra of FeMOFs and small sized CuO nanoparticles (sCuO) showed considerable overlap with the ECL emission spectra of P-C3N4 appropriately. Therefore, FeMOFs with high specific surface area were prepared and well combined with sCuO to effectively dual-quenching the ECL emission of P-C3N4 based on resonance energy transfer. Hence, a new type ECL-RET couple made up of P-C3N4-CoPdNPs (donor) and FeMOFs-sCuO (acceptor) were developed for the first time. A certain amount of P-C3N4-CoPdNPs, Ab1, BSA, NSE were modified layer by layer onto the electrode surface. Then FeMOFs-sCuO-Ab2 bioconjugates was incubated through the immune recognition binding. As a result, a sandwich-type ECL biosensor was manufactured successfully for NSE immunoassay. Under optimal experimental conditions, the limit of detection (LOD) and the limit of quantitation (LOQ) of the prepared ECL sensor for NSE analysis was 20.4 fg mL-1 and 7.99 fg mL-1, respectively, with the relative standard deviation (RSD) of 1.68%. The linear detection range was 0.0000500-100 ng mL-1. The studied immunosensor had satisfactory sensitivity, specificity and reproducibility, manifesting the suggested sensing strategy might offer a good technical means and theoretical basis for the sensitivity analysis of NSE and has a potential application in clinical diagnosis analysis.

Tumor-derived extracellular vesicles (tdEVs) are one of the most promising biomarkers for liquid biopsy-based cancer diagnostics, owing to the expression of specific membrane proteins of their cellular origin. The investigation of epithelial-to-mesenchymal transition (EMT) in cancer using tdEVs is an alternative way of evaluating the risk of malignancy transformation. An ultra-sensitive selection and detection methodology is an essential step in developing a tdEVs-based cancer diagnostic device. In this study, we developed an indium-tin-oxide (ITO) sensor integrated microfluidic device consisting of two main parts: 1) a multi-orifice flow-fractionation (MOFF) channel for extraction of pure EVs by removing blood cellular debris, and 2) an ITO sensor coupled with a geometrically activated surface interaction (GASI) channel for enrichment and quantification of tdEV. The microfluidic channel and the ITO sensors are assembled with a 3D printed magnetic housing to prevent sample leakage and to easily attach/detach the sensors to/from the microfluidic channel. The tdEVs were successfully captured on the specific antibody modified ITO surfaces in the integrated microfluidic channel. The integrated sensors showed an excellent linear response between 103 and 109 tdEVs/mL. Simultaneous evaluation of the epithelial and mesenchymal markers on the tdEV surfaces successfully revealed the EMT index of the corresponding pancreatic cancer cells. Our ITO sensor integrated microfluidic device showed excellent detection in the clinically relevant tdEVs-concentration range for patients with pancreatic cystic neoplasms. Hence, this system is expected to open a new avenue for liquid biopsy-based cancer prognostics and diagnostics.

As an essential biomarker associated with various diseases, Uracil-DNA Glycosylase (UDG) detection is vital for disease diagnosis, treatment selection, and prognosis assessment. In recent years, the signal amplification effect of the CRISPR-Cas12a trans-cleaved single-stranded DNA probe has provided an available strategy for constructing highly sensitive biosensors. However, its superior trans-cleavage activity has become a "double-edged sword" for building biosensors that can amplify the target signal while also amplifying the leakage signal, causing out of control. Therefore, the construction of structurally simple, extremely low-background, highly sensitive CRISPR-Cas12a-based biosensors is an urgent bottleneck problem in the field. Here, we applied CRISPR-Cas12a with a DNA hybridization reaction to develop a simple, rapid, low background, and highly sensitive method for UDG activity detection. It has no PAM restriction and the detection limit is as low as 2.5 × 10-6 U/mL. As far as we know, this method is one of the most sensitive methods for UDG detection. We also used this system to analyze UDG activity in tumor cells (LOD: 1 cell/uL) and to evaluate the ability to screen for UDG inhibitors. Furthermore, we verified the possibility of intracellular UDG activity imaging by transfecting the biosensors to the cells. We believe this novel sensor has good clinical application prospects and will effectively broaden the application space of CRISPR-Cas12a.

Owing to its ability to isolate single cells and perform high-throughput sorting, droplet sorting has been widely applied in several research fields. Compared with flow cytometry, droplet allows the encapsulation of single cells for cell secretion or lysate analysis. With the rapid development of this technology in the past decade, various droplet sorting devices with high throughput and accuracy have been developed. A droplet sorter with the highest sorting throughput of 30,000 droplets per second was developed in 2015. Since then, increased attention has been paid to expanding the possibilities of droplet sorting technology and strengthening its advantages over flow cytometry. This review aimed to summarize the recent progress in droplet sorting technology from the perspectives of device design, detection signal, actuating force, and applications. Technical details for improving droplet sorting through various approaches are introduced and discussed. Finally, we discuss the current limitations of droplet sorting for single-cell studies along with the existing gap between the laboratory and industry and provide our insights for future development of droplet sorters.

Diabetes and its complications are seriously threatening the health and well-being of hundreds of millions of people. Glucose levels are essential indicators of the health conditions of diabetics. Over the past decade, concerted efforts in various fields have led to significant advances in glucose monitoring technology. In particular, the rapid development of continuous glucose monitoring (CGM) based on electrochemical sensing principles has great potential to overcome the limitations of self-monitoring blood glucose (SMBG) in continuously tracking glucose trends, evaluating diabetes treatment options, and improving the quality of life of diabetics. However, the applications of minimally invasive electrochemical CGM sensors are still limited owing to the following aspects: i) invasiveness, ii) short lifespan, iii) biocompatibility, and iv) calibration and prediction. In recent years, the performance of minimally invasive electrochemical CGM systems (CGMSs) has been significantly improved owing to breakthrough developments in new materials and key technologies. In this review, we summarize the history of commercial CGMSs, the development of sensing principles, and the research progress of minimally invasive electrochemical CGM sensors in reducing the invasiveness of implanted probes, maintaining enzyme activity, and improving the biocompatibility of the sensor interface. In addition, this review also introduces calibration algorithms and prediction algorithms applied to CGMSs and describes the application of machine learning algorithms for glucose prediction.

Study of spatial and temporal aspects of signaling between individual cells is essential in understanding development, the immune response, and host-pathogen interactions. We present an automated high-throughput microfluidic platform that chemically stimulates immune cells to initiate cytokine secretion, and controls the formation of signal gradients that activate neighboring cell populations. Furthermore, our system enables controlling the cell type and density based on distance, and retrieval of cells from different regions for gene expression analysis. Our device performs these tasks in 192 independent chambers to simultaneously test different co-culture conditions. We demonstrate these capabilities by creating various cellular communication scenarios between macrophages and fibroblasts in vitro. We find that spatial distribution of macrophages and heterogeneity in cytokine secretion determine spatiotemporal gene expression responses. Furthermore, we describe how gene expression dynamics depend on a cell's distance from the signaling source. Our device addresses key challenges in the study of cell-to-cell signaling, and provides high-throughput and automated analysis over a wide range of co-culture conditions.

The hydrogen-based economy will require not only sustainable hydrogen production but also sensitive and cheap hydrogen sensors. Commercially available H2 sensors are limited by either use of noble metals or elevated temperatures. In nature, hydrogenase enzymes present high affinity and selectivity for hydrogen, while being able to operate in mild conditions. This study aims at evaluating the performance of an electrochemical sensor based on carbon nanomaterials with immobilised hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus for H2 detection. The effect of various parameters, including the surface chemistry, dispersion degree and amount of deposited carbon nanotubes, enzyme concentration, temperature and pH on the H2 oxidation are investigated. Although the highest catalytic response is obtained at a temperature around 60 °C, a noticeable current can be obtained at room temperature with a low amount of protein less than 1 μM. An original pulse-strategy to ensure H2 diffusion to the bioelectrode allows to reach H2 sensitivity of 4 μA cm-2 per % H2 and a linear range between 1 and 20%. Sustainable hydrogen was then produced through dark fermentation performed by a synthetic bacterial consortium in an up-flow anaerobic packed-bed bioreactor. Thanks to the outstanding properties of the A. aeolicus hydrogenase, the biosensor was demonstrated to be quite insensitive to CO2 and H2S produced as the main co-products of the bioreactor. Finally, the bioelectrode was used for the in situ measurement of H2 produced in the bioreactor in steady-state.

Microfluidics provides a powerful platform for biological analysis by harnessing the ability to precisely manipulate fluids and microparticles with integrated microsensors. Here, we introduce an imaging and impedance cell analyzer (IM2Cell), which implements single cell level impedance analysis and hydrodynamic mechanical phenotyping simultaneously. For the first time, IM2Cell demonstrates the capability of multi-stress level mechanical phenotyping. Specifically, IM2Cell is capable of characterizing cell diameter, three deformability responses, and four electrical properties. It presents high-dimensional information to give insight into subcellular components such as cell membrane, cytoplasm, cytoskeleton, and nucleus. In this work, we first validate imaging and impedance-based cell analyses separately. Then, the two techniques are combined to obtain both imaging and impedance data analyzed by machine learning method, exhibiting an improved prediction accuracy from 83.1% to 95.4% between fixed and living MDA-MB-231 breast cancer cells. Next, IM2Cell demonstrates 91.2% classification accuracy in a mixture of unlabeled MCF-10A, MCF-7, and MDA-MB-231 cell lines. Finally, an application demonstrates the potential of IM2Cell for the deformability studies of peripheral blood mononuclear cells (PBMCs) subpopulations without cumbersome isolation or labeling steps.

Carbapenemase-producing Gram-negative bacilli (CPGNB) is a type of antibiotic-resistant pathogens that often lead to severe clinical consequences. Phenotypic tests, such as Carba NP and blue Carba, are able to detect the resistant mechanism and provide rapid detection of carbapenemase producers to potentially guide personalized therapy. However, these tests require relatively tedious hands-on fluidic operations, and the assay format is ill-suited for automation and parallelization for improved throughput. In this study, we report an automated magnetic digital microfluidics-based platform, known as DropCarba, for parallel CPGNB detection in droplets. It automates the entire carbapenemase testing process and eliminates the need for almost all hands-on fluidic operations, which ensures high consistency and minimizes human errors with a simple "press-and-go" operation. DropCarba was validated with a large number of bacterial isolates of various Enterobacterales species (200 strains in total with 100 CPGNB and 100 non-resistant strains) in a blinded manner, and the results agree well with the benchmark Carba NP. DropCarba, with its full automation, simple operation, reduced reagent consumption, parallelization processing, and scalable manufacturing, will greatly improve CPGNB screening and make a valuable contribution to our fight against antibiotic resistance.

Rapid detection of diquat (DQ) is essential in clinical diagnosis and rescue. Here, we developed a fast, simple-yet-practical detection strategy for the reliable identification and quantification of DQ in biological fluids. Based on surface-enhanced Raman spectroscopy (SERS), point-of-care detection was realized under the acidic condition with gold nanoparticles as the substrate. Under optimal experimental conditions, the detection limits of the strategy were 17.5 ppb and 1.99 ppm in human urine and gastric juice, respectively. High specificity and selectivity of the SERS strategy were demonstrated using common pesticides and coexisting biological substances. The method was also used to detect biofluids from 5 patients and urine samples from 10 healthy volunteers. The results were in high agreement with spectrophotometric and clinical liquid chromatography-mass spectrometry methods. The volume of urine samples required for this technique is merely 20 μL, and no preparation of the samples is required. Compared to traditional methods used in clinical settings, SERS-based methods are capable of real-time measurements that accurately provide rapid detection and response in non-laboratory settings, with great potential for on-site and point-of-care testing.

In this work, PtPd nanoparticles functionalized porphyrin metal-organic framework nanoenzymes (PtPd@PCN-224 nanoenzymes) are exploited as signal amplification tags to fabricate a rapid and ultrasensitive sensitive CRISPR/Cas14a-based electrochemical biosensor for Burkholderia pseudomallei (B. pseudomallei) specific DNA sequences detection. The prepared PtPd@PCN-224 nanoenzymes not only catalyze the reduction peak current of H2O2 to obtain a strong electrochemical signal output, but also provide massive active sites for the assembly of nucleic acids by Zr-O-P bonds. Besides, the designed target-activated CRISPR/Cas14a is able to recognize the target DNA sequences and further trigger the trans-cleavage of ssDNA for signal amplification. Benefiting from the target-activated CRISPR/Cas14a and PtPd@PCN-224 nanoenzymes, the developed electrochemical biosensor for B. pseudomallei DNA detection exhibits high sensitivity with detection of limit down to 12.8 aM and excellent specificity for distinguishing non-targeted bacteria. Moreover, the CRISPR/Cas14a-based electrochemical detection platform can also apply for other pathogenic bacteria diagnostic by well-designing sgRNA for target sequence recognition, possessing high flexibility and versatility in clinical diagnosis.

Growing studies focusing on nuclear acid detection via the emerging CRISPR technique demonstrate its promising application. However, limited works solve the identification of non-nucleic acid targets, especially multiple small molecules. To address challenges for point-of-care testing (POCT) in complex matrices for healthcare, environment, and food safety, we developed CRISPR Cas12a-powered highly sensitive, high throughput, intelligent POCT (iPOCT) for multiple small molecules based on a smartphone-controlled reader. As a proof of concept, aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), and capsaicin (CAP) were chosen as multiple targets. First, three antigens were preloaded in independent microwells. Then, the antibody/antigen-induced fluorescent signals were consecutively transferred from the biotin-streptavidin to CRISPR/Cas12a system. Third, the fluorescent signals were recorded by a smartphone-controlled handheld dark-box readout. Under optimization, detection limits in AFB1, BaP, and CAP were 0.00257, 4.971, and 794.6 fg/mL with wide linear ranges up to four orders of magnitude. Using urine, water, soybean oil, wheat, and peanuts as the complex matrix, we recorded high selectivity, considerable recovery, repeatability, and high consistency comparison to HPLC-MS/MS methods. This work promises a practical intelligent POCT platform for multiple targets in lipid-soluble and water-soluble matrices and could be extensively applied for healthcare, environment, and food safety.

Although CRISPR-based biosensors for pathogenic detection are highly specific, they not sensitive enough and nucleic acid amplification is generally required to improve their sensitivity. However, this allows only binary operations and significantly limits practical applications. Here, a CRISPR/Cas12a-powered Evanescent wAve fluorescence nanobiosensing plaTform (CREAT) was developed for ultrasensitive nucleic acid amplification-free quantitative detection of pathogens with multiple signal enhancements. In addition to collateral cleavage amplification of the CRISPR/Cas12a system, we constructed nanophotonic structure-based evanescent wave fluorescence enhancement, Mg2+ or DNA-mediated fluorescence enhancement, and air-displacement fluorescence enhancement strategies for ultrasensitive detection of Staphylococcus aureus (S. aureus). Especially, the fluorescence signal detected by CREAT can be significantly enhanced by adding a simple air displacement step, thus improving detection sensitivity. This nanobiosensor detected real samples containing S. aureus, with a detection limit of 592 CFU/mL and 13.2 CFU/mL in 45 min and 90 min, respectively, which are comparable to those of RT-qPCR. This paves a new way for simple, rapid, sensitive, robust, and flexible on-site detection of S. aureus as well as other pathogens.

Aggregation of β-amyloid (Aβ) were considered as a typical pathological feature of Alzheimer's disease (AD). Extensive studies have verified that soluble Aβ oligomers (AβO) were more toxic to neurons than plaques. Herein, in this work, a glucose entrapped liposome-based portable aptasensor was fabricated for recognizing and interacting with AβO by specific aptamer on liposome (G-Lip-Apt). Then, a single strand DNA, designed to be partially complementary to AβO aptamer, was modified on amino-functionalized Fe3O4@SiO2 to obtain a magnetic nanocomposite (Fe3O4@SiO2/NH2-DNA). In the presence of AβO, the specific recognition between AβO and its aptamer on G-Lip-Apt made AβO bounded with G-Lip-Apt. With subsequent introduction of Fe3O4@SiO2/NH2-DNA, the unreacted G-Lip-Apt was further linked with Fe3O4@SiO2/NH2-DNA by double stranded complementary pairing interaction. Along with the addition of TritonX-100 into the formed G-Lip-Apt/Fe3O4@SiO2/NH2-DNA complex, the encapsulated glucose was released from liposome and then measured by a personal glucose meter (PGM). Good linear correlation was acquired over concentration of 5.0-1000 nM and the limit of detection (LOD) was calculated to be 2.27 nM for AβO. The developed portable electrochemical strategy integrated magnetic separation, competitive reaction and point of care test (POCT) to achieve high sensitivity, selectivity and accuracy, therefore enabled it successfully applied to the analysis of AβO in the hippocampus and cortex of APP/PS1 transgenic AD mice.

A highly stretchable and tissue-adhesive multifunctional sensor based on structurally engineered islets embedded in ultra-soft hydrogel is reported for monitoring of bladder activity in overactive bladder (OAB) induced rat and anesthetized pig. The use of hydrogel yielded a much lower sensor modulus (1 kPa) compared to that of the bladder (300 kPa), while the strong adhesiveness of the hydrogel (adhesive strength: 260.86 N/m) allowed firm attachment onto the bladder. The change in resistance of printed liquid metal particle thin-film lines under strain were used to detect bladder inflation and deflation; due to the high stretchability and reliability of the lines, surface strains of 200% could be measured repeatedly. Au electrodes coated with Platinum black were used to detect electromyography (EMG). These electrodes were placed on structurally engineered rigid islets so that no interfacial fracture occurs under high strains associated with bladder expansion. On the OAB induced rat, stronger signals (change in resistance and EMG root-mean-square) were detected near intra-bladder pressure maxima, thus showing correlation to bladder activity. Moreover, using robot-assisted laparoscopic surgery, the sensor was placed onto the bladder of an anesthetized pig. Under voiding and filling, bladder strain and EMG were once again monitored. These results confirm that our proposed sensor is a highly feasible, clinically relevant implantable device for continuous monitoring OAB for diagnosis and treatment.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Lateral flow immunoassay (LFIA) with gold nanoparticles (AuNPs) as the signal reporter is widely used for the rapid detection of food-borne pathogens. However, it is difficult for LFIA to achieve sensitive detection due to the insufficient colorimetric signal brightness of AuNPs. Herein, we developed a bimetallic Ag-Au urchin-like hollow nanospheres (BUHNPs) based on the simple and rapid synthesis through co-reduction and galvanic replacement reactions. The BUHNPs exhibit superb colorimetric signal brightness, strong photothermal signals and high antibody coupling efficiency. The obtained colorimetric and photothermal LFIA (BUHNPs-CM-LFIA and BUHNPs-PT-LFIA) based on BUHNPs were used for the sensitive detection of a pathogenic bacterium (Escherichia coli O157:H7). The limit of detection values of the colorimetric and photothermal quantitative analysis were 2.48 × 103 and 5.50 × 102 CFU mL-1, which were approximately 4-fold and 18-fold lower than that of AuNPs-LFIA (9.92 × 103 CFU mL-1), respectively. This work suggests that a dual-readout LFIA with BUHNPs as a promising signal reporter can be used to improve the detection performance of LFIA and construct a more accurate and sensitive detection platform.

Three-dimensional (3D) heterotypic multicellular spheroid models play important roles in researches of the proliferation and remodeling phases in wound healing. This study aimed to develop a sessile drop array to cultivate 3D spheroids and simulate wound healing stage in vitro using NIH-3T3 fibroblasts and M2-type macrophages. By the aid of the offset of surface tension and gravity, the sessile drop array is able to transfer cell suspensions to spheroids via the superhydrophobic surface of each microwell. Meanwhile, each microwell has a cylinder hole at its bottom that provides adequate oxygen to the spheroid. It demonstrated that the NIH-3T3 fibroblast spheroid and the 3T3 fibroblast/M2-type macrophage heterotypic multicellular spheroid can form and maintain physiological activities within nine days. In order to further investigate the structure without destroying the entire spheroid, we reconstructed its 3D morphology using transparent processing technology and the Z-stack function of confocal microscopy. Additionally, a nano antibody-based 3D immunostaining assay was used to analyze the proliferation and differentiation characteristics of these cells. It found that M2-type macrophages were capable of promoting the differentiation of 3T3 fibroblast spheroid. In this study, a novel, inexpensive platform was constructed for developing spheroids, as well as a 3D immunofluorescence method for investigating the macrophage-associated wound healing microenvironment.

The electrochemical biosensor with outstanding sensitivity and low cost is regarded as a viable alternative to current clinical diagnostic techniques for various disease biomarkers. However, their actual analytical use in complex biological samples is severely hampered due to the biofouling, as they are also highly sensitive to nonspecific adsorption on the sensing interfaces. Herein, we have constructed a non-fouling electrochemical biosensor based on antifouling peptides and the electroneutral peptide nucleic acid (PNA), which was used as the recognizing probe for the specific binding of the viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Different from the negatively charged DNA probes that will normally weaken the biosensors' antifouling capabilities owing to the charge attraction of positively charged biomolecules, the neutral PNA probe will generate no side-effects on the biosensor. The biosensor demonstrated remarkable sensitivity in detecting SARS-CoV-2 viral RNA, possessing a broad linear range (1.0 fM - 1.0 nM) and a detection limit down to 0.38 fM. Furthermore, the sensing performance of the constructed electrochemical biosensor in human saliva was nearly similar to that in pure buffer, indicating satisfying antifouling capability. The combination of PNA probes with antifouling peptides offered a new strategy for the development of non-fouling sensing systems capable of assaying trace disease biomarkers in complicated biological media.

Esophageal cancer is a malignant tumor with two-thirds of patients having a local recurrence or distant metastasis. To date, diagnostic biomarkers with high sensitivity and specificity are lacking. Extracellular vesicles (EVs) have shown their potential values as disease biomarkers as they carry specific proteins and RNAs derived from cancer cells. In this study, we investigate ESCC precision diagnostics from the insights of circulating EVs, and integrate the ultrafast EV isolation approach (EXODUS) and ELISA for fast detection and screening of ESCC patients. First, we isolate and characterize the high-purity plasma EVs with EXODUS and identify 401 proteins and 372 proteins from ESCC patient and healthy individuals, respectively. Further looking into the differentially expressed proteins (DEPs) of ESCC patients and enriched KEGG pathways, we discover EV-CD14 as a potential diagnostic biomarker for ESCC, which has been further validated as a significantly differentially expressed protein by Western Blot and immunogold labelling TEM. For fast screening and detection of ESCC towards clinical applications, we apply ELISA method to diagnose ESCC from 60 clinical samples based on circulating EV-CD14, which shows a high AUC value up to 96.0% for detection of ESCC in a test set (30 samples), and displays a high accuracy rate up to 90% for prediction of ESCC in a screening test (30 samples). Our results suggest that the circulating EV-CD14 may highly be related to the initiation and progression of ESCC, providing a novel method for the diagnosis and prognosis of ESCC towards clinical translations.

It is a significant challenge to perform painless invasive detection of inflammation progression in relation to the evolution of pH, redox potential, and reactive oxygen species (ROS) levels in an in situ and real-time manner. In this work, polydopamine-modified, silver nanoparticle-decorated poly (methyl methacrylate) microneedles (AgNPs@PDA@MNs) have been developed as a multiplexed surface enhanced Raman scattering (SERS) diagnostic platform. Using rhodamine 6G as the Raman signal molecule, the AgNPs@PDA@MNs demonstrated a significant enhancement with reasonable linearity in the range of 10-3-10-9 mol/L and the limit of detection is 1.0 × 10-10 mol/L 4-mercaptobenzoic acid, anthraquinone-2-carboxylic acid and para-aminothiophenol were covalently anchored on AgNPs@PDA@MNs SERS substrate. I1143/I1183, I1606/I1667 and I1143/I1077 were used as assay standards for pH, redox potential and ROS level detection, respectively. The SERS multiplexed transparent microneedles (SERS mtMNs) linearly responded to pH in the range of 4.0-8.0, redox potential in the range of 417.0-599.8 mV, and ROS levels in the range of 0-480 ng/mL, demonstrating a significant ability to detect complex inflammation in vivo, in situ and in real-time.

Gel-sol transition-based biosensors are a promising and popular alternative for portable, cost-effective, and user-friendly point-of-care testing (POCT). However, the improvement of sensitivity and practicability is highly demanded. In this work, a Fe-NC single-atom catalyst (SAC) is successfully synthesized and used as a signal amplification element for highly sensitive gel-sol transition-based biosensing. The Fe-NC SAC owns excellent peroxidase-like activity of 188 U/mg due to its definite atomically active centers and maximum atomic utilization of active metal atoms. As a proof-of-concept, the Fe-NC SAC is uniformly encapsulated in gelatin hydrogel to obtain a hydrogel sensor that allows colorimetric detection of trypsin based on gel-sol transition. The gelatin hydrogel network collapses derived from the hydrolysis by trypsin, and thereby the released Fe-NC SAC leads to the colorimetric sensing process. The designed hydrogel sensor offers a low detection limit of 1 ng/mL with a range from 1 to 100 ng/mL toward trypsin detection, exhibiting excellent selectivity and sensitivity, and well-performed practical detection in human serum. This work offers a successful paradigm for designing a promising SACs-related detection strategy and paves a new way to develop high-performance gel-sol transition-based sensors and various POCT applications.

Because of the brain's complexity, developing effective treatments for neurological disorders is a formidable challenge. Research efforts to this end are advancing as in vitro systems have reached the point that they can imitate critical components of the brain's structure and function. Brain-on-a-chip (BoC) was first used for microfluidics-based systems with small synthetic tissues but has expanded recently to include in vitro simulation of the central nervous system (CNS). Defining the system's qualifying parameters may improve the BoC for the next generation of in vitro platforms. These parameters show how well a given platform solves the problems unique to in vitro CNS modeling (like recreating the brain's microenvironment and including essential parts like the blood-brain barrier (BBB)) and how much more value it offers than traditional cell culture systems. This review provides an overview of the practical concerns of creating and deploying BoC systems and elaborates on how these technologies might be used. Not only how advanced biosensing technologies could be integrated with BoC system but also how novel approaches will automate assays and improve point-of-care (PoC) diagnostics and accurate quantitative analyses are discussed. Key challenges providing opportunities for clinical translation of BoC in neurodegenerative disorders are also addressed.

Point-of-care testing (POCT) platforms for microRNA (miRNA) detection have attracted considerable attention in recent years, due to the increasingly important role of miRNA as biomarkers for the diagnosis of many diseases, such as cancers. However, several limitations such as the requirement of enzyme-related amplification system, expensive preservation cost, sophisticated analysis instruments and tedious operations of conventional miRNA biosensing devices severely hinder their widespread applications. In this work, a portable and smart colorimetric analysis platform was developed by employing the ultrathin DNA-gold nanoparticle (AuNP) hybrid hydrogel film as the signaling unit and the enzyme-free entropy-driven dynamic DNA network (EDN) as the signal converter and amplification unit. By programming the DNA sequences of the EDN, the EDN could respond to a specific miRNA, with miRNA-155 or miRNA-21 as the model target, and release a converter DNA with amplified concentration to further trigger the release of AuNPs from the hydrogel film as a colorimetric signal output. To avoid the use of sophisticated spectral instruments, digital analysis based on primary three-color channel (R/G/B) was further introduced by using user-friendly camera and image processing software, and a detection limit at pM level was achieved. Moreover, by introducing H2O2-mediated AuNPs enlargement procedure in the colorimetric analysis platform, the detection limit for miRNA target could further be enhanced to fM level. The POCT platform is also portable and storable with a good storage stability for at least 45 days, suggesting its great potential in practical diagnosis applications.

The microRNA21 (miR-21), a specific tumor biomarker, is crucial for the diagnosis of several cancer types, and investigation of its overexpression pattern is important for cancer diagnosis. Herein, we report a low-cost, rapid, ultrasensitive, and convenient biosensing strategy for the detection of miR-21 using a nanoplasmonic array chip coupled with gold nanoparticles (AuNPs). This sensing platform combines the surface plasmon resonance effect of nanoplasmonics (NanoSPR) and the localized surface plasmon resonance (LSPR) effect, which allows the real-time monitoring of the subtle optical density (OD) changes caused by the variations in the dielectric constant in the process of the hybridization of the target miRNA. Using this method, the miRNA achieves a broad detection range from 100 aM to 1 μM, and with a limit of detection (LoD) of 1.85 aM. Furthermore, this assay also has a single-base resolution to discriminate the highly homologous miRNAs. More importantly, this platform has high throughput characteristics (96 samples can be detected simultaneously). This strategy exhibits more than 86.5 times enhancement in terms of sensitivity compared to that of traditional biosensors.

An ultrasensitive electrochemical immunosensor based on signal amplification of the deposition of the electroactive ferrocene-tyramine (Fc-Tyr) molecule, catalyzed by horseradish peroxidase (HRP), was constructed for the detection of the liver cancer marker Glypican-3 (GPC3). Functional electroactive molecule Fc-Tyr is reported to exhibit both the enzymatic cascade catalytic activity of tyramine signal amplification (TSA) and the excellent redox properties of ferrocene. In terms of design, the low matrix effects inherent in using the magnetic bead platforms, a quasi-homogeneous system, allowed capturing the target protein GPC3 without sample pretreatment, and loading HRP to trigger the TSA, which induced a large amount of Fc-Tyr deposited on the electrode surface layer by layer as a signal probe for the detection of GPC3. The concept of Fc-Tyr as an electroactive label was validated, GPC3 biosensor exhibited high selectivity and sensitivity to GPC3 in the range of 0.1 ng mL-1-1 μg mL-1. Finally, the sensor was used simultaneously with ELISA to assess GPC3 levels in the serum of clinical liver cancer patients, and the results showed consistency, with a recovery of 98.33-105.35% and a relative standard deviation (RSD) of 4.38-8.18%, providing a theoretical basis for achieving portable, rapid and point of care testing (POCT) of tumor markers.

Nowadays, the studies on the interaction and relationship between the intestinal microorganisms and liver diseases are increasing. However, it is still a huge challenge for the in-depth investigation and dynamic monitoring of such a complex network. Herein, a significant discovery was made. A strong association between gut microbial structural and functional genomics and SERS spectra of hepatocytes were revealed. Based on the study of gut microbes and SERS spectra, complementary information could be provided for the mechanism analysis of related diseases. Liver fibrosis, a chronic liver disease that lack specific cure was thus comprehensive studied. Liver targeting gold nanoparticle dimers were prepared as the SERS tags, and abundant SERS peak signals were acquired. Meanwhile, the gut microbiomes were also comparative studied. The changes of carbohydrates and lipids in liver cells were observed at the early stages of liver fibrosis, and TLR4 (toll-like receptors 4) was activated to elicit immune responses. Then again, oxidative stress, endotoxin and serum inflammatory factors were the major observations at the late stages. The SERS signals and the microbiome analysis were well confirmed and complemented each other, which suggested that the detection strategy could be another valuable method for the "gut-liver axis" study.

Real-time monitoring in the tumor microenvironment provides critical insights of cancer progression and mechanistic understanding of responses to cancer treatments. However, clinical challenges and significant questions remain regarding assessment of limited clinical tissue samples, establishment of validated, controllable pre-clinical cancer models, monitoring of static versus dynamic markers, and the translation of insights gained from in vitro tumor microenvironments to systematic investigation and understanding in clinical practice. State-of-art tumor-on-a-chip strategies will be reviewed herein, and emerging real-time sensing and monitoring platforms for on-chip analysis of tumor microenvironment will also be examined. The integration of the sensors with tumor-on-a-chip platforms to provide spatiotemporal information of the tumor microenvironment and the associated challenges will be further evaluated. Though optimal integrated systems for in situ monitoring are still in evolution, great promises lie ahead that will open new paradigm for rapid, comprehensive analysis of cancer development and assist clinicians with powerful tools to guide the diagnosis, prognosis and treatment course in cancer.