The preservation of nucleus structure during microscopy imaging is a top priority for understanding chromatin organization, genome dynamics, and gene expression regulation. In this review, we summarize the sequence-specific DNA labelling methods that can be used for imaging in fixed and/or living cells without harsh treatment and DNA denaturation: (i) hairpin polyamides, (ii) triplex-forming oligonucleotides, (iii) dCas9 proteins, (iv) transcription activator-like effectors (TALEs) and (v) DNA methyltransferases (MTases). All these techniques are capable of identifying repetitive DNA loci and robust probes are available for telomeres and centromeres, but visualizing single-copy sequences is still challenging. In our futuristic vision, we see gradual replacement of the historically important fluorescence in situ hybridization (FISH) by less invasive and non-destructive methods compatible with live cell imaging. Combined with super-resolution fluorescence microscopy, these methods will open the possibility to look into unperturbed structure and dynamics of chromatin in living cells, tissues and whole organisms.

In addition to carrying and transmitting genetic material, some DNA molecules have specific binding ability or catalytic function. DNA with this special function is collectively referred to as functional DNA (fDNA), such as aptamer, DNAzyme and so on. fDNA has the advantages of simple synthetic process, low cost and low toxicity. It also has high chemical stability, recognition specificity and biocompatibility. In recent years, fDNA biosensors have been widely investigated as signal recognition elements and signal transduction elements for the detection of non-nucleic acid targets. However, the main problem of fDNA sensors is their limited sensitivity to trace targets, especially when the affinity of fDNA to the targets is low. To further improve the sensitivity, various nucleic acid signal amplification strategies (NASAS) are explored to improve the limit of detection of fDNA. In this review, we will introduce four NASAS (hybridization chain reaction, entropy-driven catalysis, rolling circle amplification, CRISPR/Cas system) and the corresponding design principles. The principle and application of these fDNA sensors integrated with signal amplification strategies for detection of non-nucleic acid targets are summarized. Finally, the main challenges and application prospects of NASAS integrated fDNA biosensing system are discussed.

Portable and on-site detection of target biomarker is of great significance in early diagnosis of diseases. Herein, we designed a portable smartphone-based PEC immunoassay platform to detect prostate specific antigen (PSA) adopting Co-doped Bi2O2S nanosheets as photoactive materials. The fast photocurrent response under visible light and excellent electrical transport rate invest Co-doped Bi2O2S with the property of being effectively excited even under a weak light source. Therefore, with the incorporation of a carriable flashlight that act as the excitation light source, disposable screen-printed electrodes, a microelectrochemical workstation and a smartphone that served as control center, point-of-care analytical detection of low-abundance small molecule analytes was successfully realized. Specifically, a sandwich-type immunoreaction was performed using alkaline phosphatase labeled secondary antibody as signal indicator. In the presence of PSA, ascorbic acid as generated through a catalytic reaction, resulting in the enhancement of photocurrent intensity. The photocurrent intensity increased linearly with the logarithm of PSA concentrations ranging from 0.2 to 50 ng mL-1 with a detection limit of 71.2 pg mL-1 (S/N = 3). This system provided an effective method for the construction of portable and miniaturized PEC sensing platform for the application of point-of-care health monitoring.

Protein-nucleic acid interactions are not only fundamental to genetic regulation and cellular metabolism, but molecular basis to artificial biosensors. However, such interactions are generally weak and dynamic, making their specific and sensitive quantitative detection challenging. By using primer exchange reaction (PER)-amplified protein-nucleic acid interactions, we here design a universal and ultrasensitive electrochemical sensor to quantify microRNAs (miRNAs) in blood. This PER-miR sensor leverages specific recognition between S9.6 antibodies and miRNA/DNA hybrids to couple with PER-derived multi-enzyme catalysis for ultrasensitive miRNA detection. Surface binding kinetic analysis shows a rational Kd (8.9 nM) between the miRNA/DNA heteroduplex and electrode-attached S9.6 antibody. Based on such a favorable affinity, the programmable PER amplification enables the sensor to detect target miRNAs with sensitivity up to 90.5 aM, three orders of magnitude higher than that without PER in routine design, and with specificity of single-base resolution. Furthermore, the PER-miR sensor allows detecting multiple miRNAs in parallel, measuring target miRNA in lysates across four types of cell lines, and differentiating tumor patients from healthy individuals by directly analyzing the human blood samples (n = 40). These advantages make the sensor a promising tool to enable quantitative sensing of biomolecular interactions and precision diagnostics.

Using biosensor to screen for Alzheimer's disease (AD) facilitates early detection of AD with high sensitivity and accuracy. This approach overcomes the limitations of conventional AD diagnostic methods, such as neuropsychological assessment and neuroimaging analysis. Here, we propose a simultaneous analysis of signal combinations generated by four crucial AD biomarkers (Amyloid beta 1-40 (Aβ40), Aβ42, total tau 441 (tTau441), and phosphorylated tau 181 (pTau181)) by inducing a dielectrophoretic (DEP) force on fabricated interdigitated microelectrode (IME) sensor. By applying an optimal DEP force, our biosensor selectively concentrates and filters the plasma-based AD biomarkers, exhibiting high sensitivity (limit of detection <100 fM) and selectivity in the plasma-based AD biomarkers detection (p < 0.0001). Consequently, it is demonstrated that a complex combined signal comprising four AD-specific biomarker signals (Aβ40- Aβ42+ tTau441- pTau181) can differentiate between patients with AD and healthy subjects with high accuracy (78.85%) and precision (80.95%) (p < 0.0001).

Herein, we presented a dual-readout gasochromic immunosensing platform for accurate and sensitive detection of carcinoembryonic antigen (CEA) based on Ag-doped/Pd nanoparticles loaded MoO3 nanorods (Ag/MoO3-Pd). Initially, the presence of analyte CEA would prompt the formation of sandwich-type immunoreaction, accompanied by the introduction of Pt NPs labeled on detection antibody. Upon the addition of NH3BH3, the product hydrogen (H2) will interact with Ag/MoO3-Pd as a bridge between the sensing interface and the biological assembly platform. Both photocurrent and temperature signals can serve as readouts due to the significantly increased PEC performance and enhanced photothermal conversion capability of H-Ag/MoO3-Pd (the product of Ag/MoO3-Pd react with H2) compared to Ag/MoO3-Pd. In addition, the DFT results show that the band gap of Ag/MoO3-Pd becomes narrower after the reaction with H2, thus improving the utilization of light, which theoretically explains the internal mechanism of gas sensing reaction. Under optimal conditions, the designed immunosensing platform showed good sensitivity for CEA detection with the limit of detection (LOD) of 26 pg mL-1 (photoelectrochemical mode) and 98 pg mL-1 (photothermal mode). This work not only presents the possible reaction mechanism of Ag/MoO3-Pd and H2, but also creatively applicate it in photothermal biosensors that give a new path for devising dual-readout immunosensor.

It is essential to construct a biofuel cell-based sensor and develop an effective strategy to detect glucose without any potentiostat circuitry in order to create a simple and miniaturized device. In this report, an enzymatic biofuel cell (EBFC) is fabricated by the facile design of an anode and cathode on a screen-printed carbon electrode (SPCE). To construct the anode, thionine and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) are covalently immobilized via a crosslinker to make a cross-linked redox network. As a cathode, the Pt-free oxygen reduction carbon catalyst is employed alternative to the commonly used bilirubin oxidase. We proposed the importance of EBFC-based sensors through the connection of anode and cathode; they can identify a short-circuit current by means of applied zero external voltage, thereby capable of glucose detection without under the operation of the potentiostat. The result shows that the EBFC-based sensor could be able to detect based on a short-circuit current with a wide range of glucose concentrations from 0.28 to 30 mM. Further, an EBFC is employed as a one-compartment model energy harvester with a maximum power density of (36 ± 3) μW cm- 2 in sample volume 5 μL. In addition, the constructed EBFC-based sensor demonstrates that the physiological range of ascorbic acid and uric acid shows no significant effect on the short-circuit current generation. Moreover, this EBFC can be used as a sensor in artificial plasma without losing its performance and thereby used as a disposable test strip in real blood sample analysis.

The CRISPR/Cas system is known as one of the directions of the next generation of mainstream molecular diagnostic technology. However, most current CRISPR/Cas molecular diagnostics still rely on the pre-amplification of nucleic acid due to the limited sensitivity of CRISPR/Cas alone, which has no significant advantage over commercial Taqman-PCR and TwistAmp® Exo kits. Herein, we report an aM-level sensitive cascade CRISPR-Dx system (ASCas) that eliminates nucleic acid pre-amplification, thus avoiding aerosol contamination and greatly reducing the testing environment and personnel skill requirements for molecular diagnostics. Most importantly, the Cas13a nucleases with high sensitivity and trans-cleavage efficiency can rapidly cleaved RNA bubbles on the hybridized cascade probe at low concentration target RNA detection, which results in the destruction of the cascade probe and releases a large amount of trigger DNA for further signal amplification of secondary Cas12a reactions. Therefore, the ASCas system achieves amplification-free, ultra-sensitivity (1 aM), and ultra-fast (20 min) RNA detection. In addition, the ASCas system replaces the complicated screening process of primers and probes with the programmed Cas13a-crRNA design so that a suitable detection system can be constructed more quickly and straightforwardly for the mutation-prone SARS-CoV-2 virus.

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL-1. The detection limit was defined as 4.3 CFU mL-1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

This work demonstrates an organic electrochemical transistor (OECT) immuno-sensor with a detection limit down to fg mL-1. The OECT device transforms the antibody-antigen interaction signal by using the zeolitic imidazolate framework-enzyme-metal polyphenol network nanoprobe, which can produce electro-active substance (H2O2) through the enzyme-catalytic reaction. The produced H2O2 is subsequently electrochemically oxidized at the platinum loaded CeO2 nanosphere-carbon nanotube modified gate electrode, resulting in an amplified current response of the transistor device. This immuno-sensor realizes the selective determination of vascular endothelial growth factor 165 (VEGF165) down to the concentration of 13.6 fg mL-1. It also shows good applicable capacity for determining the VEGF165 that human brain microvascular endothelial cells and U251 human glioblastomas cells secreted in the cell culture medium. The ultrahigh sensitivity of the immuno-sensor is derived from excellent performances of the nanoprobe for enzyme loading and the OECT device for H2O2 detection. This work may provide a general way to fabricate the OECT immuno-sensing device with high performances.

To develop highly accurate and ultrasensitive strategies is of great importance for the clinical measurement, in particular, the detection of cancer biomarkers. Herein, we synthesized an ultrasensitive TiO2/MXene/CdS QDs (TiO2/MX/CdS) heterostructure as a photoelectrochemical immunosensor, which favors energy levels matching and fast electron transfer from CdS to TiO2 in the help of ultrathin MXene nanosheet. Dramatic photocurrent quenching can be observed upon incubation of the TiO2/MX/CdS electrode by Cu2+ solution from 96-well microplate, which caused by the formation of CuS and subsequent CuxS (x = 1, 2), reducing the absorption of light and boosting the electron-hole recombination upon irradiation. As a result, the as-prepared biosensor demonstrates a linearly increased photocurrent quenching percentage (Q%) value with CEA concentration ranging from 1 fg/mL to 10 ng/mL, as well as a low detection limit of 0.24 fg/mL. Benefit from its excellent stability, high selectivity and good reproducibility of as-prepared PEC immunosensor, we believe that this proposed strategy might provide new opportunities for clinical diagnosis of CEA and other tumor markers.

Capturing, identifying, and counting CTCs cancer cells that have escaped from the tumor and wandered into the bloodstream is a major challenge. We proposed a noval microswimmer dual-mode aptamer (electrochemical and fluorescent)-(Mapt-EF) homogeneous sensor with active capture/controlled release double signaling molecule/separation and release cell based on the Co-Fe-MOF nanomaterial for simultaneous one-step detection of multiple biomarkers protein tyrosine kinase-7 (PTK7), Epithelial cell adhesion molecule (EpCAM), and mucin-1 (MUC1) for diagnosis of multiple cancer cell types. The Co-Fe-MOF is a nano-enzyme capable of catalyzing the decomposition of hydrogen peroxide to release bubbles of oxygen, producing a driving force to conduct hydrogen peroxide through the liquid, and has the capacity to self-decompose during the catalytic process. Phosphoric acid is present in the aptamer chains of PTK7, EpCAM, and MUC1, and the aptamer chains are adsorbed to the surface of the Mapt-EF homogeneous sensor in the form of a gated switch to inhibit the catalytic decomposition activity of hydrogen peroxide. The Mapt-EF homogeneous sensor has the capability to actively target biomarkers that can be entrained by oxygen bubbles without being degraded. The detection time of the sensor was 20 min, the detection limits were 9.6 fg/mL, 8.4 fg/mL and 7.7 fg/mL with the linear range was 0-20 pg/mL, respectively. The Mapt-EF homogeneous sensor has high detection sensitivity, and its detection limit can reach the level of single cell at the lowest. The Mapt-EF homogeneous sensor has great application potential in clinical detection and analysis of tumor cells.

Chronic sampling of tonic serotonin (5-hydroxytryptamine, 5-HT) concentrations in the brain is critical for tracking neurological disease development and the time course of pharmacological treatments. Despite their value, in vivo chronic multi-site measurements of tonic 5-HT have not been reported. To fill this technological gap, we batch-fabricated implantable glassy carbon (GC) microelectrode arrays (MEAs) onto a flexible SU-8 substrate to provide an electrochemically stable and biocompatible device/tissue interface. To achieve detection of tonic 5-HT concentrations, we applied a poly(3,4-ethylenedioxythiophene)/carbon nanotube (PEDOT/CNT) electrode coating and optimized a square wave voltammetry (SWV) waveform for selective 5-HT measurement. In vitro, the PEDOT/CNT-coated GC microelectrodes achieved high sensitivity to 5-HT, good fouling resistance, and excellent selectivity against the most common neurochemical interferents. In vivo, our PEDOT/CNT-coated GC MEAs successfully detected basal 5-HT concentrations at different locations within the CA2 region of the hippocampus of both anesthetized and awake mice. Furthermore, the PEDOT/CNT-coated MEAs were able to detect tonic 5-HT in the mouse hippocampus for one week after implantation. Histology reveals that the flexible GC MEA implants caused less tissue damage and reduced inflammatory response in the hippocampus compared to commercially available stiff silicon probes. To the best of our knowledge, this PEDOT/CNT-coated GC MEA is the first implantable, flexible sensor capable of chronic in vivo multi-site sensing of tonic 5-HT.

Fumonisin B1 (FB1), the most prevalent and highest toxicity mycotoxins among fumonisins family, poses threats to human especially children and infants even at a trace level. Therefore, its facile and sensitive detection is of importance. Herein, Z-scheme Cu2MoS4/CdS/In2S3 nanocage-like heterojunctions (labeled Cu2MoS4/CdS/In2S3) were synthesized, whose photoelectrochemical (PEC) property and electron transfer mechanism were strictly investigated. The Cu2MoS4/CdS/In2S3 behaved as photoactive substrate for building a PEC sensing platform for detection of FB1, integrated with PtPd alloy modified hollow CoSnO3 nanoboxes (labeled PtPd-CoSnO3) nanozyme. By virtue of the stronger affinity between the target FB1 and its aptamer (FB1-Apt), the photocurrent was recovered by releasing the CoSnO3-PtPd3 modified FB1-Apt (FB1-Apt/PtPd-CoSnO3) from the photoanode, which can terminate the catalytic precipitation reaction for its peroxidase-like property. The resultant PEC aptasensor exhibited a wider dynamic linear range from 1 × 10-4 to 1 × 102 ng mL-1 with a lower limit of detection (0.0723 pg mL-1). Thus, this research provides a feasible PEC sensing platform for routine analysis of other mycotoxins in practice.

The coral reef crisis has significantly intensified over the last decades, mainly due to severe outbreaks of crown-of-thorns starfish (COTS). Current ecological monitoring has failed to detect COTS densities at the pre-outbreak stage, thus preventing early intervention. In this work, we developed an effective electrochemical biosensor modified by a MoO2/C nanomaterial, as well as a specific DNA probe that could detect trace COTS environmental DNA (eDNA) at a lower detection limit (LOD = 0.147 ng/μL) with excellent specificity. The reliability and accuracy of the biosensor were validated against the standard methods by an ultramicro spectrophotometer and droplet digital PCR (p > 0.05). The biosensor was then utilized for the on-site analysis of seawater samples from SYM-LD and SY sites in the South China Sea. For the SYM-LD site suffering an outbreak, the COTS eDNA concentrations were 0.33 ng/μL (1 m, depth) and 0.26 ng/μL (10 m, depth), respectively. According to the ecological survey, the COTS density was 500 ind/hm2 at the SYM-LD site, verifying the accuracy of our measurements. At the SY site, COTS eDNA was also detected at 0.19 ng/μL, but COTS was not found by the traditional survey. Hence, larvae were possibly present in this region. Therefore, this electrochemical biosensor could be used to monitor COTS populations at the pre-outbreak stages, and potentially serve as a revolutionary early warning method. We will continue to improve this method for picomolar or even femtomolar detection of COTS eDNA.

To construct an electrochemical sensing interface which was convenient for protease recognition and cleavage, we designed a strategy for directed self-assembly of histidine-tagged peptides on the electrode led by Ni2+ ions for electrochemical detection of prostate specific antigen (PSA). The electrode surface was first functionalized using carboxylated multiwalled carbon nanotubes and then modified with the metal ion chelating agent (5 S)-N-(5-Amino-1-carboxypentyl) iminodiacetic acid (NIA). After the Ni2+ was captured by NIA, the designed immune-functional peptide could be oriented assembly to the electrode interface through the imidazole ring of histidine at the tail, completing the construction of the recognition layer. Therefore, by adding the analyte PSA to identify and shear the immune-functional peptide, the ferrocene in its head was released, resulting in a reduction in the electrical signal, enabling sensitive detection. In addition, the self-assembly layer could be removed by pickling to realize the reconstruction of the recognition layer. Under optimal conditions, the electrochemical sensor had an ultralow detection limit of 11.8 fg mL-1 for PSA, with a wide detection range from 1 pg mL-1 to 100 ng mL-1. In this work, an electrochemical sensing interface based on the histidine-tagged peptide induced by Ni2+ was formed to enable controllable oriented assembly on the electrode surface, and the recognition layer could be reconstructed via pickling, providing a potential approach for the design of repeatable interfaces.

Alzheimer's disease (AD) is a neurodegenerative disease of complex pathogenesis, with overt symptoms following disease progression. Early AD diagnosis is challenging due to the lack of robust biomarkers and limited patient access to diagnostics via neuroimaging and cerebrospinal fluid (CSF) tests. Exosomes present in body fluids are attracting attention as diagnostic biomarkers that directly reflect neuropathological features within the brain. In particular, exosomal miRNAs (exomiRs) signatures are involved in AD pathogenesis, showing a different expression between patients and the healthy controls (HCs). However, low yield and high homologous nature impede the accuracy and reproducibility of exosome blood-based AD diagnostics. Here, we developed a programmable curved plasmonic nanoarchitecture-based biosensor to analyze exomiRs in clinical serum samples for accurate AD diagnosis. To allow the detection of exomiRs in serum at attomolar levels, nanospaces (e.g., nanocrevice and nanocavity) were introduced into the nanostructures to dramatically increase the spectral sensitivity by adjusting the bending angle of the plasmonic nanostructure through sodium chloride concentration control. The developed biosensor classifies individuals into AD, mild cognitive impairment (MCI) patients, and HCs through profiling and quantifying exomiRs. Furthermore, integrating analysis expression patterns of multiple exosomal biomarkers improved serum-based diagnostic performance (average accuracy of 98.22%). Therefore, precise, highly sensitive serum-derived exosomal biomarker detection-based plasmonic biosensor has a robust capacity to predict the molecular pathologic of neurodegenerative disease, progression of cognitive decline, MCI/AD conversion, as well as early diagnosis and treatment.

The release of cytokines by chimeric antigen receptor (CAR) T-cells and tumor resident immune cells defines a significant part of CAR T-cell functional activity and patient immune responses during CAR T-cell therapy. However, few studies have so far precisely characterized the cytokine secretion dynamics in the tumor niche during CAR T-cell therapy, which requires multiplexed, and timely biosensing platforms and integration with biomimetic tumor microenvironment. Herein, we implemented a digital nanoplasmonic microarray immunosensor with a microfluidic biomimetic Leukemia-on-a-Chip model to monitor cytokine secretion dynamics during CD19 CAR T-cell therapy against precursor B-cell acute lymphocytic leukemia (B-ALL). The integrated nanoplasmonic biosensors achieved precise multiplexed cytokine measurements with low operating sample volume, short assay time, heightened sensitivity, and negligible sensor crosstalk. Using the digital nanoplasmonic biosensing approach, we measured the concentrations of six cytokines (TNF-α, IFN-γ, MCP-1, GM-CSF, IL-1β, and IL-6) during first 5 days of CAR T-cell treatment in the microfluidic Leukemia-on-a-Chip model. Our results revealed a heterogeneous secretion profile of various cytokines during CAR T-cell therapy and confirmed a correlation between the cytokine secretion profile and the CAR T-cell cytotoxic activity. The capability to monitor immune cell cytokine secretion dynamics in a biomimetic tumor microenvironment could further help in study of cytokine release syndrome during CAR T-cell therapy and in development of more efficient and safer immunotherapies.

Ultrasensitive determination of tumor marker (TM) is of great significance in cancer prevention and diagnosis. Traditional TM detection methods involve large instrumentation and professional manipulation, which complicate the assay procedures and increase the cost of investment. To resolve these problems, an integrated electrochemical immunosensor based on the flexible polydimethylsiloxane/gold (PDMS/Au) film with Fe-Co metal-organic framework (Fe-Co MOF) as a signal amplifier was fabricated for ultrasensitive determination of alpha fetoprotein (AFP). First, gold layer was deposited on the hydrophilic PDMS film to form the flexible three-electrode system, and then the thiolated aptamer for AFP was immobilized. Afterward, the aminated Fe-Co MOF possessing high peroxidase-like activity and large specific surface area was prepared by a facile solvothermal method, and subsequently the biofunctionalized MOF could effectively capture biotin antibody (Ab) to form MOF-Ab as a signal probe and amplify the electrochemical signal remarkably, thereby realizing highly sensitive detection of AFP with a wide linear range of 0.01-300 ng/mL and a low detection limit of 0.71 pg/mL. In addition, the PDMS based-immunosensor showed good accuracy for assaying of AFP in clinical serum samples. The integrated and flexible electrochemical immunosensor based on the Fe-Co MOF as a signal amplifier demonstrates great potential for application in the personalized point-of-care (POC) clinical diagnosis.

MicroRNA-125b (miR-125b) is highly associated with synaptic dysfunction and tau hyperphosphorylation in the early pathogenesis of Alzheimer's disease (AD), making it a promising biomarker for early AD diagnosis. Hence, there is an urgent need for a reliable sensing platform to assist in situ miR-125b detection. In this work, we report a dual "turn-on" fluorescence biosensor based on the nanocomposite of aggregation-induced emission fluorogen (AIEgen)-labeled oligonucleotide (TPET-DNA) probes immobilized on the surface of cationic dextran modified molybdenum disulfide (TPET-DNA@Dex-MoS2). In the presence of the target, TEPT-DNA can hybridize with miR-125b to form a DNA/RNA duplex, causing TPET-DNA to detach from the surface of Dex-MoS2 that simultaneously activates the dual fluorescence enhancement processes: (1) recovery of TPET-DNA signal and (2) strong fluorescent emission from AIEgen triggered by restriction of the intramolecular rotation. The sensing performance of TPET-DNA@Dex-MoS2 was demonstrated by detecting miR-125b in vitro with good sensitivity at the picomolar level and rapid response (≤1 h) without amplification procedures. Furthermore, our nanoprobes exhibited excellent imaging capabilities to aid real-time monitoring of the endogenous miR-125b in PC12 cells and brain tissues of mice AD model induced by local administration of okadaic acid (OA). The fluorescence signals of the nanoprobes indicated miR-125b was spatially associated with phosphorylated tau protein (p-tau) in vitro and in vivo. Therefore, TPET-DNA@Dex-MoS2 could be a promising tool for in situ and real-time monitoring of the AD-related microRNAs and also provide mechanistic insight into the early prognosis of AD.

A relatively new approach to subcellular research is Raman microscopy with the application of sensors called Raman probes. This paper describes the use of the sensitive and specific Raman probe, 3-O-propargyl-d-glucose (3-OPG), to track metabolic changes in endothelial cells (ECs). ECs play a significant role in a healthy and dysfunctional state, the latter is correlated with a range of lifestyle diseases, particularly with cardiovascular disorders. The metabolism and glucose uptake may reflect the physiopathological conditions and cell activity correlated with energy utilization. To study metabolic changes at the subcellular level the glucose analogue, 3-OPG was used, which shows a characteristic and intense Raman band at 2124 cm-1.3-OPG was applied as a sensor to track both, its accumulation in live and fixed ECs and then metabolism in normal and inflamed ECs, by employing two spectroscopic techniques, i.e. spontaneous and stimulated Raman scattering microscopies. The results indicate that 3-OPG is a sensitive sensor to follow glucose metabolism, manifested by the Raman band of 1602 cm-1. The 1602 cm-1 band has been called the "Raman spectroscopic signature of life" in the cell literature, and here we demonstrate that it is attributed to glucose metabolites. Additionally, we have shown that glucose metabolism and its uptake are slowed down in the cellular inflammation. We showed that Raman spectroscopy can be classified as metabolomics, and its uniqueness lies in the fact that it allows the analysis of the processes of a single living cell. Gaining further knowledge on metabolic changes in the endothelium, especially in pathological conditions, may help in identifying markers of cellular dysfunction, and more broadly in cell phenotyping, better understanding of the mechanism of disease development and searching for new treatments.

A desirable lanthanide-based ratiometric fluorescent probe was designed and integrated into a self-designed Fermat spiral microfluidic chip (FS-MC) for the automated determination of a unique bacterial endospore biomarker, dipicolinic acid (DPA), with high selectivity and sensitivity. Here, a blue emission wavelength at 425 nm was generated in the Fermat spiral structure by mixing the europium (Eu3+) and luminol to form the Eu3+/Luminol sensing probe. DPA in the reservoir can be used to specifically bind to Eu3+ under the negative pressure and transfer energy from DPA to Eu3+ sequentially via an antenna effect, thus resulting in a significant increase in the red fluorescence emission peak at 615 nm. According to the fluorescence intensity ratio (F615/F425), a good linearity can be obtained with increasing the concentration of DPA from 0 to 200 μM with a limit of detection as low as 10.11 nM. Interestingly, the designed FS-MC can achieve rapid detection of DPA in only 1 min, reducing detection time and improving sensitivity. Furthermore, a self-designed device integrated with the FS-MC and a smartphone color picker APP was employed for the rapid automatic point-of-care testing (POCT) of DPA in the field, simplifying complex processes and reducing testing times, thus confirming the great promise of this ready-to-use measurement platform for in situ inspection.

Artificial intelligence (AI) has received great attention since the concept was proposed, and it has developed rapidly in recent years with applications in many fields. Meanwhile, newer iterations of smartphone hardware technologies which have excellent data processing capabilities have leveraged on AI capabilities. Based on the desirability for portable detection, researchers have been investigating intelligent analysis by combining smartphones with AI algorithms. Various examples of the application of AI algorithm-based smartphone detection and analysis have been developed. In this review, we give an overview of this field, with a particular focus on bioanalytical detection applications. The applications are presented in terms of hardware design, software algorithms, and specific application areas. We also discuss the existing limitations of AI-based smartphone detection and analytical approaches, and their future prospects. The take-home message of our review is that the application of AI in the field of detection analysis is restricted by the limitations of the smartphone's hardware as well as the model building of AI for detection targets with insufficient data. Nevertheless, at this juncture, while bioanalytical diagnostics and health monitoring have set the pace for AI-based smartphone applicability, the future should see the technology making greater inroads into other fields. In relation to the latter, it is likely that the ordinary or average person will play a greater participatory role.

Antimonite (SbIII) is a naturally occurring contaminant demanding on-site ultrasensitive detection. The enzyme-based electrochemical (EC) biosensors are promising, but the lack of specific SbIII oxidizing enzymes hindered the past efforts. Herein, we modulated the specificity of arsenite oxidase AioAB toward SbIII by regulating its spatial conformation from tight to loose using the metal-organic framework ZIF-8. The constructed EC biosensor, AioAB@ZIF-8, exhibited the substrate specificity toward SbIII at 12.8 s-1 μM-1, an order of magnitude higher than that of AsIII (1.1 s-1 μM-1). Relaxing AioAB structure in ZIF-8 was evidenced by the break of the S-S bond and the conversion of α helix to the random coil as suggested by Raman spectroscopy. Our AioAB@ZIF-8 EC sensor exhibited a dynamic linear range in 0.041-4.1 μM at a response time of 5 s, and the detection limit at 0.041 μM at a high sensitivity of 1894 nA μM-1. The insights into tuning the specificity of an enzyme shed new light on biosensing metal(loid)s without specific proteins.

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect a characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposure assessment.

Optimized and sensitive biomarker detection has recently been shown to have a critical impact on quality of diagnosis and medical care options. In this research study, polyoxometalate-γ-cyclodextrin metal-organic framework (POM-γCD MOF) was utilized as an electrocatalyst to fabricate highly selective sensors to detect in-situ released dopamine. The POM-γCD MOF produced multiple modes of signals for dopamine including electrochemical, colorimetric, and smartphone read-outs. Real-time quantitative monitoring of SH-SY5Y neuroblastoma cellular dopamine production was successfully demonstrated under various stimuli at different time intervals. The POM-CD MOF sensor and linear regression model were used to develop a smartphone read-out platform, which converts dopamine visual signals to digital signals within a few seconds. Ultimately, POM-γCD MOFs can play a significant role in the diagnosis and treatment of various diseases that involve dopamine as a significant biomarker.

Currently, only a few small devices are capable of continuously recording the physiological states of neurons in real time. Micro-electrode arrays (MEAs) are widely used as electrophysiological technology to detect the excitability of neurons non-invasively. However, the development of miniaturized and multi-parameter MEAs capable of real-time recording remains challenging. In this study, an on-chip micro-electrode and platinum resistor array (MEPRA) biosensor was designed and fabricated to monitor both the electrical and temperature signals of cells synchronously in real time. Such on-chip sensor maintains high sensitivity and stability. The MEPRA biosensor was further used to investigate the effects of propionic acid (PA) on primary neurons. The results demonstrate that PA affects the temperature and firing frequency of primary cortical neurons in concentration-dependent manners. The changes of temperature and firing frequency work in tandem with neuronal physiological status, including neuron viability, intracellular calcium concentration, neural plasticity, and mitochondrial function. This highly biocompatible, stable, and sensitive MEPRA biosensor may provide high-precision reference information for investigating the physiological responses of neuron cells under various conditions.

This work develops a novel photoelectrochemical sensor for the detection of carcinoembryonic antigen (CEA) based on the composite of UCNPs with semiconductors and conformational changes in the DNA structure. Firstly, SnS2, ZnIn2S4 and UCNPs were assembled on the surface of the ITO electrode. Then Au NPs were dropped, which could facilitate the coupling of CdSe NPs modified DNA1 via Au-S bond, giving an ITO/SnS2/ZnIn2S4/UCNPs/CdSe heterojunction structure. When irradiated with 980 nm near-infrared (NIR) light, the UV-visible light emitted by the UCNPs could excite the nanocomposite, producing an enhanced photoelectric reaction. Subsequently, CEA aptamer and DNA2-modified SiO2 were added to form a Y-shaped DNA structure. At this time, the photocurrent was significantly reduced by the combination of the light-blocking effect of SiO2 and the departure of CdSe NPs from the electrode surface. When the target CEA was added, the recognition between CEA and the aptamer led to the collapse of the Y-shaped DNA structure, the restoration of hairpin DNA and the proximity of CdSe to the electrode. Accordingly, the photocurrent signals enhanced again. Under optimal experimental conditions, the detection limit as low as 0.3 pg mL-1 was obtained with good selectivity, achieving a sensitive "on-off-on" photoelectrochemical sensor for CEA detection.

Magnetic separation was often applied to isolate and concentrate foodborne bacteria using immunomagnetic nanobeads before downstream bacterial detection. However, nanobead-bacteria conjugates (magnetic bacteria) were coexisting with excessive unbound nanobeads, limiting these nanobeads on magnetic bacteria to further act as signal probes for bacterial detection. Here, a new microfluidic magnetophoretic biosensor was elaboratively developed using a rotated high gradient magnetic field and platinum modified immunomagnetic nanobeads for continuous-flow isolation of magnetic bacteria from free nanobeads, and combined with nanozyme signal amplification for colorimetric biosensing of Salmonella. First, the platinum modified immunomagnetic nanobeads were mixed with the bacterial sample to form the magnetic bacteria, and magnetically separated to eliminate non-magnetic background. Then, the mixture of free immunomagnetic nanobeads and magnetic bacteria was injected with sheath flow (PBS) at higher flowrate into the semi-circle magnetophoretic separation channel under rotated magnetic field, which was generated by two repulsive cylindric magnets and their in-between ring iron gear, leading to continuous-flow isolation of magnetic bacteria from free immunomagnetic nanobeads because they suffered from different magnetic forces and thus had different deviating positions at the outlet. Finally, the separated magnetic bacteria and unbound magnetic nanobeads were respectively collected and used to catalyze coreless substrate into blue product, which was further analyzed using the microplate reader to obtain bacterial amount. This biosensor could determinate Salmonella as low as 41 CFU/mL in 40 min.

Lipid droplets (LDs) are critical organelles involved in many physiological processes in eukaryotic cells. To visualize and study LDs, particular the small/nascent LDs, the emerging super-resolution fluorescence imaging techniques with nanoscale resolution would be much more powerful in comparison to the conventional confocal/wide-field imaging techniques. However, directly limited by the availability of advanced LDs probes, super-resolution fluorescence imaging of LDs is a practically challenging task. In this context, a superior LDs fluorescent probe named Lipi-Deep Red is newly developed for structured illumination microscopy (SIM) super-resolution imaging. This fluorescent probe features with the advantages of strong deep red/NIR emission, fluorogenic character, high LDs specificity, and outstanding photostability. These advantages enable the fluorescent probe to be finely applied in SIM super-resolution imaging, e.g. time-lapse imaging (up to 1000 frames) to monitor the LDs dynamics at nanoscale (159 nm), two-color time-lapse imaging to discover the nearby contact/interaction between LDs and mitochondria. Consequently, the fusion processes of LDs are impressively visualized at a high spatial and temporal resolution. Two kinds of contact models between LDs and mitochondria (dynamic contact and stable contact) newly proposed in the recent literatures are successfully revealed.

Portable devices for on-site foodborne pathogens detection are urgently desirable. Lateral flow immunoassay (LFIA) provides an efficient strategy for pathogens detection, however, antibody labeling independence and detection reliability, are still challenging. Here, we report the development of a label-free LFIA with dual-readout using glucan-functionalized two-dimensional (2D) transition metal dichalcogenides (TMDs) tungsten disulfide (WS2) as detection probes for sensitive detection of Salmonella enteritidis (S. enteritidis). In particular, glucan-functionalized WS2, synthesized via liquid exfoliation, are reliable detection antibody candidates which served as antibody mimics for bacteria capturing. This LFIA has not only eliminated the intricate antibody labeling process and screening of paired antibodies in conventional LFIAs, but also promised dual-readout (colorimetric/Raman) for flexible detection. Under optimized conditions, this LFIA achieves selective detection of S. enteritidis with a low visual detection limit of 103 CFU/mL and a broad linear range of 103-108 CFU/mL. Additionally, the LFIA could be successfully applied in drinking water and milk with recoveries of 85%-109%. This work is desirable to expand the application of 2D TMDs in biosensors and offers a brand-new alternative protocol of detection antibodies in foodborne pathogens detection.

The diagnosis of periprosthetic joint infection (PJI) remains a labor-intensive and challenging issue, with life-threatening complications associated with misdiagnoses. Superior diagnostic approaches are therefore urgently needed, and synovial biomarkers are gaining substantial attention in this capacity. A new aptamer-based sandwich assay was developed where the aptamer probes specific to one such biomarker, alpha-defensin human neutrophil protein 1 (HNP 1), was integrated herein into a new microfluidic platform. The magnetic beads coated with the primary aptamer probe were able to bind the target protein with high affinity and high specificity in synovial fluid and a fluorescent-labelled secondary aptamer were further used to quantify HNP 1 in a sandwich approach. Up to four clinical samples with low volume (∼50 μL each) in a much faster assay including detection within <60 min with 100% accuracy (with totally 13 clinical samples without the need of sample pretreatment) through the use of the aptamer-based sandwich assay were automatically detected on a single chip. The wide dynamic range of this compact device, 0.5-100 mg/L, highlights its utility for future PJI diagnostics in the clinic.

Sensitive quantification of multiple analytes of interest is of great significance for clinical diagnosis. CRISPR Cas platforms offer a strategy for improving the specificity, sensitivity, and speed of nucleic acid-based diagnostics, while their multiplex analysis capability is still limited and challenging. Herein, we develop a novel DNA Tetrahedron (DTN)-supported biosensor based on the spatially separated CRISPR Cas self-amplification strategy and multiple-metal-nanoparticle tagging coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection to improve the sensitivity and feasibility of the platform for multiplex detection of HPV-DNA (HPV-16, HPV-18 and HPV-52). Given target DNA induces robust trans-cleavage activity of the Cas12a/crRNA duplex, and the surrounding corresponding single-stranded DNA (ssDNA) linker are cleaved into short fragments that are unable to bond metal-nanoparticle probes (197Au, 107Ag, 195Pt) onto DTN modified magnetic beads probe (MBs-DTN), resulting in obvious ICP-MS signal change. Of note, compared with ssDNA functionalized MBs, a higher Signal-to-Noise Ratio was obtained by using MBs-DTN in our system, further amplifying the signal by regulating probes on the surface of MBs. As expected, the HPV-DNA could be detected with detection limits as low as 218 fM and be multiplexed assayed at one test with high accuracy and specificity by this proposed strategy. Furthermore, we demonstrated that the HPV-DNA in cervical swab samples could be detected, showing high consistency with DNA sequencing results. We believe that this work provides a promising option in designing CRISPR based multiplex detection system for high sensitivity, good specificity, and clinical molecular diagnostics.

Rapid, sensitive, and inexpensive point-of-care diagnosis is vital to controlling highly infectious diseases, including COVID-19. Here, we report the design and characterization of a compact fluorimeter called a "Virus Pod" (V-Pod) that enables sensitive self-testing of SARS-CoV-2 viral load in saliva. The rechargeable battery-operated device reads the fluorescence generated by Designer DNA Nanostructures (DDN) when they specifically interact with intact SARS-CoV-2 virions. DDNs are net-shaped self-assembling nucleic acid constructs that provide an array of highly specific aptamer-fluorescent quencher duplexes located at precise positions that match the pattern of spike proteins. The room-temperature assay is performed by mixing the test sample with DNA Net sensor in a conventional PCR tube and placing the tube into the V-Pod. Fluorescent signals are generated when multivalent aptamer-spike binding releases fluorescent quenchers, resulting in rapid (5-min) generation of dose-dependent output. The V-Pod instrument performs laser excitation, fluorescence intensity quantitation, and secure transmission of data to an App via Bluetooth™. We show that the V-Pod and DNA Net assay achieves clinically relevant detection limits of 3.92 × 103 viral-genome-copies/mL for pseudo-typed wild-type SARS-CoV-2 and 1.84 × 104, 9.69 × 104, 6.99 × 104 viral-genome-copies/mL for pathogenic Delta, Omicron, and D614G variants, representing sensitivity similar to laboratory-based PCR. The pocket-sized instrument (∼$294), inexpensive reagent-cost/test ($1.26), single-step, rapid sample-to-answer, and quantitative output represent a capability that is compatible with the needs of frequent self-testing in a consumer-friendly format that can link with medical service systems such as healthcare providers, contact tracing, and infectious disease reporting.

Two-electrode (2E) system of the interdigitated electrode array (IDA), which operates neither reference nor counter electrodes, has great potential to miniaturize multiplex immunoassay in a microfluidic chip for point-of-care testing. However, it is necessary to firmly immobilize the mediator layer on IDA made of indium tin oxide (ITO) which is chemically inert. It is important because the mediator determines the electrochemical potential in the 2E system, but the layer is easy to be detached during the washing processes of immunoassay. Here, we controlled the concentration of ethylenediamine (EDA) to generate a permeable and robust film to adhere to mediators on the ITO IDA chip. Electrooxidation of EDA yielded thin oligomeric ethyleneimine (OEI) film and it provided amine groups for immobilizing the mediator, poly(toluidine blue) (pTB), via common conjugation reaction. Despite repeated flows in the microchannel, which are essential for sensitive immunoassay, the pTB/OEI layer was hardly washed and still remained on the ITO IDA. Myoglobin was measured down to ∼ pg/mL level. Therefore, the ITO IDA modified with the OEI film in the 2E system constituted a stable platform that withstands washing steps for sensitive electrochemical detection in the miniaturized immunoassay.

Antibody assay for SARS-CoV-2 has become increasingly important to track latent and asymptomatic infections, check the individual's immune status, and confirm vaccine efficacy and durability. However, current SARS-CoV-2 antibody assays require invasive blood collection, requiring a remote laboratory and a trained phlebotomist. Direct detection of SARS-CoV-2 antibodies from clinical saline gargle samples has been considered challenging due to the smaller number of antibodies in such specimens and the high limit of detection of currently available rapid tests. This work demonstrates simple and non-invasive methods for detecting SARS-CoV-2 salivary antibodies. Competitive particle immunoassays were developed on a paper microfluidic chip using the receptor-binding domain (RBD) antigens on spike proteins. Using a smartphone, they were monitored by counting the captured fluorescent particles or evaluating the capillary flow velocities. The limit of detection (LOD), cross-binding between alpha- and omicron-strains, and the effect of angiotensin-converting enzyme 2 (ACE2) presence were investigated. LODs were 1-5 ng/mL in both 10% and 1% saliva. Clinical saline gargle samples were assayed using both methods, showing a statistical difference between virus-negative and virus-positive samples, although the assays targeted antibodies. Only a small number of virus-positive samples were antibody-negative. The high assay sensitivity detected a small number of antibodies developed even during the early phase of infections. Overall, this work demonstrates the ability to detect SARS-CoV-2 salivary IgG antibodies on simple, cost-effective, portable platforms towards mitigating SARS-CoV-2 and potentially other respiratory viruses.

The morphological and mechanical characteristics of red blood cells (RBCs) largely vary depending on the occurrence of hematologic disorders. Variations in the rheological properties of RBCs affect the dynamic motions of RBCs, especially their rotational behavior. However, conventional techniques for measuring the orientation of biconcave-shaped RBCs still have some technical limitations, including complicated optical setups, complex post data processing, and low throughput. In this study, we propose a novel image-based technique for measuring 3D position and orientation of normal RBCs using digital in-line holographic microscopy (DIHM) and artificial intelligence (AI). Formaldehyde-fixed RBCs are immobilized in coagulated polydimethylsiloxane (PDMS). Holographic images of RBCs positioned at various out-of-plane angles are acquired by precisely manipulating the PDMS-trapped RBC sample attached to a 4-axis optical stage. With the aid of deep learning algorithms for data augmentation and regression analysis, the out-of-plane angle of RBCs is directly predicted from the captured holographic images. The 3D position and in-plane angle of RBCs are acquired by employing numerical reconstruction and ellipse detection methods. Combining these digital image processing techniques, the 3D positional and orientational information of each RBC recorded in a single holographic image is measured within 23.5 and 3.07 s, respectively. The proposed AI-based DIHM technique that can extract the 3D position, orientation, and morphology of individual RBCs would be utilized to analyze the dynamic translational and rotational motions of abnormal RBCs with hematologic disorders in shear flows through further research.

In this study, an all-in-one poly(methyl methacrylate) (PMMA) device integrating two novel techniques - DNA extraction employing a CuSO4/H2O2 system and DNA detection utilizing solid phase copper tape - coupled with loop-mediated isothermal amplification (LAMP) is developed for on-site pathogen detection. The CuSO4/H2O2 system, also known as Fenton-like reaction, is used to produce hydroxyl radicals, which can disrupt bacterial membranes via lipid peroxidation and release DNA at room temperature. The released DNA is subsequently amplified by LAMP reaction. The acidic environment resulting from the production of hydrogen ions in the presence of target DNA in the LAMP reaction can stimulate the color change on copper tape due to the corrosion, while the innate alkaline environment in a negative sample not containing target DNA cannot stimulate the corrosion. The fabricated PMMA device integrates all the functionalities necessary for molecular diagnostics such as DNA extraction, amplification, and detection, and a carbon paste-based heater is fabricated for LAMP reaction. Using the PMMA device, Enterococcus faecium was detected as low as 4.67 × 102 CFU/mL within 90 min. E. faecium spiked in milk was successfully detected using the all-in-one PMMA device. The equipment-free techniques for decentralized diagnostics and naked-eye readout of results coupled with the portable heater serves as a promising solution for point-of-care testing particularly in a resource-limited environment.

Simultaneous detection of multiple biomarkers can allow to reduce the costs of medical diagnostics, and thus improve the accuracy and effectiveness of disease diagnosis and prognosis. Here, for the first time, we present a low-cost, simple, and rapid method for simultaneous detection of three matrix metalloproteinases (MMP-1, MMP-2, and MMP-9) that play important roles in the progression of lung cancer. The sensor matrix was constructed using a G2 polyamidoamine dendrimer (PAMAM) containing amino, carboxyl, and sulfhydryl groups. The recognition process was based on specific enzymatic cleavage of the Gly-Ile peptide bond by MMP-1, Gly-Leu bond by MMP-2, and Gly-Met bond by MMP-9, and monitoring was done by square wave voltammetry. The activity of metalloproteinases was detected based on the change of current signals of redox receptors (dipeptides labeled with electroactive compounds) covalently anchored onto the electrode surface. The conditions of the biosensor construction, including the concentration of receptors on the sensor surface and the time of interaction of the receptor with the analyte, were carefully optimized. Under optimal conditions, the linear response of the developed method ranged from 1.0⋅10-8 to 1.0 mg⋅L-1, and the limit of detection for MMP-1, MMP-2, and MMP-9 was 0.35, 0.62, and 1.10 fg⋅mL-1, respectively. The constructed biosensor enabled us to efficiently profile the levels of active forms of MMP-1, MMP-2, and MMP-9 in tissue samples (plasma and lung and tumor extracts). Thus, the developed biosensor can aid in the early detection and diagnosis of lung cancer.

The continued emergence of SARS-CoV-2 variants of concern (VOCs) has raised great challenges for epidemic prevention and control. A rapid, sensitive, and on-site SARS-CoV-2 genotyping technique is urgently needed for individual diagnosis and routine surveillance. Here, a field-deployable ultrasensitive CRISPR-based diagnostics system, called Chemical additive-Enhanced Single-Step Accurate CRISPR/Cas13 Testing system (CESSAT), for simultaneous screening of SARS-CoV-2 and its five VOCs (Alpha, Beta, Gamma, Delta, and Omicron) within 40 min was reported. In this system, a single-step reverse transcription recombinase polymerase amplification-CRISPR/Cas13a assay was incorporated with optimized extraction-free viral lysis and reagent lyophilization, which could eliminate complicated sample processing steps and rigorous reagent storage conditions. Remarkably, 10% glycine as a chemical additive could improve the assay sensitivity by 10 times, making the limit of detection as low as 1 copy/μL (5 copies/reaction). A compact optic fiber-integrated smartphone-based device was developed for sample lysis, assay incubation, fluorescence imaging, and result interpretation. CESSAT could specifically differentiate the synthetic pseudovirus of SARS-CoV-2 and its five VOCs. The genotyping results for 40 clinical samples were in 100% concordance with standard method. We believe this simple but efficient enhancement strategy can be widely incorporated with existing Cas13a-based assays, thus leading a substantial progress in the development and application of rapid, ultrasensitive, and accurate nucleic acid analysis technology.