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Rapid screening for foodborne pathogens is crucial for food safety. A rapid and one-step electrochemical sensor has been developed for the detection of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella typhimurium (S. typhimurium). Through the construction of aptamer/two-dimensional carboxylated Ti3C2Tx (2D C-Ti3C2Tx)/two-dimensional Zn-MOF (2D Zn-MOF) composites, the recognition elements, signal tags, and signal amplifiers are integrated on the electrode surface. Pathogens are selectively captured using the aptamer, which increases the impedance of the electrode surface，leads to a decrease in the 2D Zn-MOF current. Bacteria can be rapidly quantified using a one-step detection method and the replacement of aptamers. The detection limits for E. coli, S. aureus, and S. typhimurium are 6, 5, and 5 CFU·mL-1, respectively. The sensor demonstrated reliable detection capabilities in real-sample testing. Therefore, the one-step sensor based on the 2D Zn-MOF and 2D C-Ti3C2Tx has significant application value in the detection of foodborne pathogens.

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Developing non-passivating and fully integrated electrode arrays for point-of-care testing of carcinoembryonic antigen (CEA) is crucial, as the serum level of CEA is closely associated with colorectal cancer. Herein, we propose a simple, low-cost, and eco-friendly template-assisted filtration method for the scalable preparation of carbon nanotube-bridged Ti3C2Tx MXene (MX@CNT) electrode arrays with a conductive network. Furthermore, we fabricate a homogeneous electrochemical (HEC) sensor for CEA detection by integrating a magnetic-bead-based alkaline phosphatase-linked immunoassay (MB-aElisa), which enables the in-situ generation of the electroactive substance 1-naphthol (1-NP). Benefiting from the unique electrochemical characteristics of a MX@CNT electrode array, such as ultra-low background signal and superior electrocatalytic activity towards the hydrolyzed 1-NP, the MB-aElisa-based HEC sensor specifically measures CEA within a detection range spanning from 0.005 to 1.0 ng mL-1, achieving a detection limit of 1.6 pg mL-1. Subsequently, this biosensing prototype is successfully utilized for the detection of CEA in serum specimens obtained from colorectal cancer patients. More importantly, the integration of MB-aElisa with a MX@CNT electrode array not only marks a significant advancement but also enables the creation of a one-step homogeneous electrochemical immunosensing platform, serving as a paradigm for the highly sensitive and selective measurement of trace tumor markers in complex biological samples.

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In general, vertical flow assay (VFA) has a disadvantage of requiring a complex analysis process that involves manually injecting various reagents (target analyte, washing buffer, detection conjugate, etc.) sequentially. However, in this study, we have developed an innovative paper-based VFA device that replaces the complex analysis process with one-step and enables the detection of multiple targets. The fabrication process of the multi-target detection VFA device is as follows: preparation and pre-treatment of the strip materials, design of strip cartridge, design of the multiple detection VFA device, optimization experiments for strip sample flow rates, determination of device analysis time, determination of device limit of detection (LOD), multiple target signal uniformity experiment, immunoglobulin G (IgG) and C-reactive protein (CRP) antigen-antibody multiple detection experiment, and data extraction and analysis method. The use of paper-based materials enables the device to be produced at cost-effective, and cartridge production allowed for uniform array formation. IgG and CRP are used to evaluate the performance of the device as common biomarkers. The device proposed in this study is currently under research. To validate multiple target detection capability of the VFA device proposed in this study, two types of antigens-antibodies (Human IgG and Human CRP) were employed. The detection limit is 0.15 µg/mL for IgG and 0.19 µg/mL for CRP in naked eye. Furthermore, it was confirmed that there is no cross-reactivity caused by the device structure through IgG and CRP antigens. In conclusion, the VFA device proposed in this study consists of a one-step analysis process, and it has been confirmed that it can detect multiple targets simultaneously.

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Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

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Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/µL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

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Food-borne pathogens in dairy products that was contaminated from raw ingredients or improper food handling can cause a major threaten to human health. Here, to construct the pathogens detection, a dual-signal readout fluorescent switching sensor was designed for one-step determination of Staphylococcus aureus (S. aureus), which was a marker of food contamination. Graphene oxide (GO) was used as a fluorescence quencher, while fluorophore-labeled hairpin DNA was used as a donor, resulting in fluorescence resonance energy transfer (FRET) from the fluorophore to GO (signal off). Enzyme-free hybridization chain reaction could generate remarkable signal amplification, which avoided the nonspecific desorption caused by any enzymatic proteins in GO surface. With the strong binding ability of aptamer to S. aureus, a long bifluorescent molecules-labeled double-stranded DNA product was formed, bringing in dual-signal readout responses (signal on). Consequently, a reliable, sensitive and selective sensor was obtained for one-step quantification of S. aureus concentration from 10 to 108 CFU/mL with a detection limit of 1 CFU/mL. Furthermore, satisfactory stability, reproducibility, specificity and good recovery efficiency in milk samples revealed that the proposed sensor could be served as a prospective tool for food safety analysis.

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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.

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CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

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With the use of DNA as building blocks, a variety of microRNA amplification-based sensing systems have been developed. Nevertheless, ultrasensitive, selective and rapid detection of microRNAs with a high signal-to-background ratio and point mutation discrimination ability remains a challenge. Herein, we propose a novel wheel drive-based DNA sensing system (NWDS) based on a self-assembled, self-quenched nanoprobe (SQP) to conduct highly specific and ultrasensitive one-step measurement of microRNAs. In this work, a signalling recognition DNA hairpin (DH) sequence with a self-complementary stem domain of 14 base pairs was used, which contained three functional regions, namely a recognition region for the target miRNA-21, a sticky region with 9 complementary nucleotides to the 3'terminus of a DNA wheel (DW) and a region for the hybridization with a quenching DNA primer (DP). The SQP was ingeniously self-assembled at room temperature by the DH and DP, which was capable of eliminating unwanted background signals. MiRNA-21 was employed as a target model to specifically activate the SQP, leading to specific hybridization between the HP and DW. With the assistance of a polymerase, an SQP-based wheel driving took place to induce hybridization/polymerization displacement cycles, initiating target recycling and DP displacement. As a result, a large amount of the newly formed hybrid SQP/DW accumulated to generate a substantially enhanced fluorescence signal. In this way, the newly proposed NWDS exhibits ultrasensitivity with a detection limit of 5.62 aM across a wide linear dynamic response range up to 200 nM, excellent selectivity with the capability to discriminate homologous miRNAs and one-base, two-base and three-base mismatched sequences, and an outstanding analytical performance in complex systems. In addition, the significant simultaneous advantages of one-step operation, rapid detection within 15 min and a high signal-to-background ratio of 26 offer a unique opportunity to promote the early diagnosis of cancer-related diseases and molecular biological analysis.

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Quantification of circulating microRNAs (miRNAs) or viral RNAs is of great significance because of their broad relevance to human health. Currently, quantitative reverse transcription polymerase chain reaction (qRT-PCR), as well as microarray and gene sequencing, are considered mainstream techniques for miRNA identification and quantitation and the gold standard for SARS-CoV2 detection in the COVID-19 pandemic. However, these laboratory techniques are challenged by the low levels and wide dynamic range (from aM to nM) of miRNAs in a physiological sample, as well as the difficulty in the implementation in point-of-care settings. Here, we describe a one-step label-free electrochemical sensing technique by assembling self-folded multi-stem DNA-redox probe structure on gold microelectrodes and introducing a reductant, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), in the detection buffer solution to achieve ultrasensitive detection with a detection limit of 0.1 fM that can be further improved if needed.

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As a major threat to food safety due to their pathogenicity, foodborne bacteria have received much attention. In this paper, we present a one-step and wash-free microfluidic biosensor platform by smartphone for simultaneous multiple foodborne bacteria target single-stranded DNA (ssDNA) detection. This technology is based on the fluorescence resonance energy transfer (FRET) between the graphene oxide (GO) and fluorescence molecules modified capture ssDNA of the target bacteria ssDNA (ctDNA) which were coated on the microfluidic chips. The fluorescence recovery was recorded by a smartphone fluorescent detector. With an optimal analytical performance, the platform realized the detection of four kinds of bacteria ssDNA simultaneously within 5 min, with the limits of detection (LODs) of 0.17, 0.18, 0.27, and 0.17 nM, respectively. And the throughput analysis of trace amounts of foodborne bacteria ssDNA in milk and water samples were successfully detected. This one-step and wash-free microfluidic biosensor can be used as a tool for food safety analysis.

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Nanotechnology has attracted much attention, and may become the key to a whole new world in the fields of food, agriculture, building materials, machinery, medicine, and electrical engineering, because of its unique physical and chemical properties, including high surface area and outstanding electrical and optical properties. The bottom-up approach in nanofabrication involves the growth of particles, and we were inspired to propose a novel nanoplasmonic method to detect the formation of nanoparticles in real time. This innovative idea may contribute to the promotion of nanotechnology development. An increase in nanometer particle size leads to optical extinction or density (OD)-value changes in our nanosensor chip at a specific wavelength measured in a generic microplate reader. Moreover, in applying this method, an ultrasensitive nanoplasmonic immunoturbidimetry assay (NanoPITA) was carried out for the high-throughput quantification of hypersensitive C-reactive protein (CRP), a well-known biomarker of cardiovascular, inflammatory, and tumor diseases. The one-step detection of the CRP concentration was completed in 10 min with high fidelity, using the endpoint analysis method. The new NanoPITA method not only produced a linear range from 1 ng/mL to 500 ng/mL CRP with the detection limit reduced to 0.54 ng/mL, which was an improvement of over 1000 times, with respect to regular immunoturbidity measurement, but was also effective in blood detection. This attractive method, combined with surface plasmon resonance and immunoturbidimetry, may become a new technology platform in the application of biological detection.

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The detection of tumor markers in blood samples with high efficiency and sensitivity is in urgent need. In this work, a one-step quantitative detection assay for alpha fetal protein (AFP) based on gold microelectrode which is denoted as AuµE through square wave voltammetry using [Fe(CN)6]3-/4- as mediator was developed. As the biorecognition element of the assay, sulfydryl-modified AFP aptamer could be directly conjugated onto the surface of the AuµE, which could capture AFP with high specificity, and this attachment would cause the decrease of the capacitive current of the cyclic voltammetry due to the reduction of the active area of the electrodes. Under the optimized conditions, the AuµE aptasensor exhibited a linear detection range for AFP from 10-10 to 10-7 g/mL (S = 7.6 nA/dec, R2 = 0.991), and the detection limit is 2.5 × 10-11 g/mL. The AuµEs aptasensor demonstrates good selectivity against other types of proteins and small molecules, and has good reproducibility. The real blood samples were used for detection of AFP using the AuµEs aptasensor, the results agree well with those provided by the hospital through electrochemiluminescence method. Herein, the proposed one-step detection assay has a great application potential in point-of-care clinical diagnostics.

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In this work, a one-step homogeneous micro-orifice resistance immunoassay has been proposed for chlorpyrifos detection by integrating functionalized polystyrene (PS) microsphere probes with particle counting technology. The particle counter is highly sensitive and accurate for detecting the state of PS microspheres, where the particles of different states exhibit significant differences in resistance. The state of the functionalized PS microspheres is altered from dispersed to aggregated during the antigen-antibody recognition. Based on the degree of aggregation of the functionalized PS microsphere probes, chlorpyrifos can be quantitatively detected through the competitive immune response between PS antibodies and PS complete antigens. This one-step homogeneous micro-orifice resistance immunoassay simplified the procedures and greatly increased the sensitivity of detection, which has been successfully applied to detect chlorpyrifos in orange samples within 0.5 h, with the detection limit of 0.058 ng/mL.

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Staphylococcus aureus (S. aureus) is a pathogen closely associated with foodborne diseases. We prepared a reliable colorimetric sensor to detect S. aureus using click chemical reaction and immunomagnetic separation. Aptamer-functionalized and ALP-labeled Fe3O4 NPs act as separation and signal transduction elements. Under the optimized conditions, the Cu+ generated by signal transduction triggers a click chemistry reaction, which causes the aggregation of azides and alkyne-AuNPs and a color change. The net extinction ratio of Δ(A530/A760) was linearly correlated with the S. aureus concentration from 10 to 106 cfu mL-1, and the limit detection was 2.4 cfu mL-1. The recoveries were 91.15 ~ 106.36% for the analysis of spiked food and water samples without pre-enrichment. Therefore, we believe that the detection platform can be easily and accurately used for S. aureus detection, providing a broad prospect for on-site visual detection.

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Three-dimensional (TD) deoxyribonucleic acid (DNA) tweezers were programmed for one-step identification and detection of ochratoxin A (OTA) and zearalenone (ZEN). The unfolding of the TD-DNA tweezers by aptamers specific to these two mycotoxins "turned" the fluorescent signals "on." The bonding of the aptamers to their corresponding targets in OTA and ZEN "turned" the fluorescent signals and the DNA tweezers "off." The detection limit of the TD-DNA tweezers for OTA and ZEN was 0.032 and 0.037 ng mL-1, respectively. The feasibility of this method was tested using two samples. Detection via this method increased the recovery of OTA and ZEN from 95.8% to 110.2%. Spike recovery and certified food products were used to detect applicability in actual situations. Analyte detection in complex samples using TD-DNA tweezers is rapid, as the process involves a single operational step. This proposed design has considerable potential for application in mycotoxin detection.

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CpG methylation is one the most predominant epigenetic modification that has been recognized as a molecular-level biomarker for various human diseases. Taking advantage of methylation-dependent cleavage and encoding flexibility in nucleic acid functions and structures, we demonstrate the cooperative in situ assembly of G-quadruplex DNAzyme nanowires for one-step sensing of CpG methylation in human genomes. This nanodevice displays good specificity and high sensitivity with a limit of detection (LOD) of 0.565 aM in vitro and 1 cell in vivo. It can distinguish 0.001% CpG methylation level from excess unmethylated DNA, quantify different CpG methylation targets from diverse human cancer cells, and even discriminate CpG methylation expressions between lung tumor and precancerous tissues. Importantly, this nanodevice can be performed isothermally in one step within 2 h in a label-free manner without any bisulfite conversion, fluorescence tagging, and PCR amplification process, providing a new platform for genomic methylation-related clinical diagnosis and biomedical research.

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The global food waste problem, especially aquatic product spoilage, stimulates the accurate freshness analysis of food products. However, it still remains a great challenge to realize in-field determination of fish freshness at the time of use. In the present study, a colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine (Hx), which is an important intermediate of adenosine triphosphate decomposition during fish storage. We demonstrate that xanthine oxidase grade I ammonium sulfate suspension (XOD-ASS) possesses peroxidase activity. It can oxidize different peroxidase substrates, including 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and o-phenylenediamine in the presence of H2O2, producing visible color reactions. Further experiments indicate that XOD-ASS displayed effective peroxidase activity and could be used for H2O2 detection. Based on this, a one-step Hx detection method was established using only XOD-ASS as the catalyst. The method displays a good linear relationship in the range from 20 to 100 µM with a detection limit of 6.93 µM. Additionally, we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage times. The recovery values range from 97.44 to 102.56%. It is exciting to note that, compared with other methods, our proposed method provides a robust advantage on the economic reaction system, ease of preparation, short time consumption, and moderate reaction temperature. We believe that this method shows good application prospects for on-site fish freshness determination.

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Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

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Early screening of L. monocytogenes in ready-to-eat food can prevent and control its harmful effects. In this study, we propose a highly sensitive magnetic DNA sensor based on nucleic acid hybridization reaction and magnetic signal readout. We design the L. monocytogenes specific probe1 and probe2 and label them on the 30 and 250 nm magnetic nanoparticles, respectively. The hybridization reaction between the magnetic probes and DNA of L. monocytogenes could form a sandwich nanocomplex. After magnetic separation, the unbound MNP30-probe2 can act as the transverse relaxation time (T2) signal readout probe. This assay allows the one-step detection of L. monocytogenes as low as 50 CFU/mL within 2 h without DNA amplification, and the average recovery in the spiked ham sausage samples can reach 92.6%. This system integrates the high sensitivity of magnetic sensing and high efficiency of hybridization reaction, providing a promising detection platform for pathogens.

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