Short Assay Time Research Articles

MicroRNA-based Biosensors for Early Detection of Cancers.

Small noncoding microRNAs (miRNAs) are known as noninvasive biomarkers for early detection in various cancers. In fact, miRNAs have key roles in carcinogenicity process such as proliferation, apoptosis and metastasis. After cardiovascular disease, cancer is the second cause of death in the world with an estimated 9.6 million deaths in 2018. So, early diagnosis of cancer is critical for successful treatment. To date, several selective and sensitive laboratory-based methods have been applied for the detection of circulating miRNA, but a simple, short assay time and low-cost method such as a biosensor method as an alternative approach to monitor cancer biomarker is required. In this review, we have highlighted recent advances in biosensors for circulating miRNA detection.

Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin.

Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.

Development of a kinetic antibody-dependent cellular cytotoxicity assay

Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism of action (MOA) of monoclonal antibody (mAb) therapeutics. Target cells opsonized with therapeutic antibody bind and activate FcγR-bearing immune effector cells, resulting in target cell lysis. A key step in mAb drug development is the characterization of ADCC activity for its potential to inform mAb efficacy and safety. A number of in vitro assays are commonly used to assess ADCC. Most are endpoint assays that measure a surrogate marker of cell lysis. Newer imaging technologies allow direct measurement of ADCC-mediated cell lysis over time. In this study, we detail the development and characterization of a kinetic ADCC assay applicable to multiple target and effector cell types. This kinetic assay shows comparable sensitivity to an endpoint fluorescence release ADCC assay, while offering the advantages of a simpler set up and shorter assay time. Our results demonstrate that kinetic ADCC activity is a valid alternative assay format for measuring in vitro ADCC of mAbs.

A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe.

The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5' hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5U·mL-1), a low detection limit (0.01U·mL-1) and short assay time (typically 60min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics. Graphical abstract A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.

Comparison of multiple detection methods of Mycoplasma pneumoniae antibody for the early diagnosis of pediatric mycoplasma pneumonia

Mycoplasma pneumoniae pneumonia (MPP) is a common disease in infants and young children. MPP can also be a potential cause of healthcare-associated pneumonia. However, it is challenging to make an accurate diagnosis in a timely fashion. Our goal is to determine the assay consistencies of acridinium ester chemiluminescence immunoassay (AECLIA) and gelatin particle agglutination (GPA) test for the detection of Mycoplasma pneumoniae (MP) antibody. In this study, a total of 1404 children with suspected MPP were enrolled. Among them, 130 were diagnosed as MPP positive by mycoplasma culture, and 186 were negative. MP antibodies were detected by AECLIA, enzyme-linked immunosorbent assay (ELISA) and GPA. Consistency rates, differences among assays, and diagnosis performance were compared for the three methods. The independent χ2 test results of AECLIA and ELISA for the detection of MP-IgG and MP-IgM antibodies were χ2 = 29.210, P < 0.001; χ2 = 9.081, P = 0.017, respectively, suggesting that the two detection methods are well correlated. Similar analyses were done for the comparison of AECLIA and GPA as well as the comparison of ELISA and GPA. The positive rates of these methods agree with epidemiology data and have good consistency. Thus, AECLIA with a much shorter assay time could be a better option for the screening of MPP.

An Immuno-Biochip Selectively Captures Tumor-Derived Exosomes and Detects Exosomal RNAs for Cancer Diagnosis.

Tumor-derived exosomes (TEXs) play instrumental roles in tumor growth, angiogenesis, immune modulation, metastasis, and drug resistance. TEX RNAs are a new class of noninvasive biomarkers for cancer. Neither current techniques, such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) and next-generation sequencing, nor new ones, such as electrochemical or surface plasmon resonance-based biosensors, are able to selectively capture and separate TEXs from normal cell-derived exosomes, making TEX RNAs potentially less sensitive biomarkers. We developed an immuno-biochip that selectively captures TEXs using antibodies against tumor-associated proteins and quantifies in situ TEX RNAs using cationic lipoplexes containing molecular beacons. We used the immuno-biochip to measure the expression of miR-21 microRNA and TTF-1 mRNA in EGFR- or PD-L1-bearing exosomes from human sera and achieved absolute sensitivity and specificity in distinguishing normal controls from non-small cell lung cancer patients. Our results demonstrated that the effective separation of TEXs from other exosomes greatly improved the detection sensitivity and specificity. Compared with the traditional immunomagnetic separation-RNA isolation-qRT-PCR workflow, the immuno-biochip showed superior lung cancer diagnostic performance, consumed less samples (∼30 μL), and shortened assay time from ∼24 to 4 h.

Enhanced Metabolic Activity of Cytochrome P450 via Carbon Nanocage-Based Photochemical Bionanoreactor.

Recently, the early screening of the genotoxicity of new chemicals and drugs calls for the envelope of micro-/nanoreactors for metabolic study. Herein, a novel light-driven enzymatic bionanoreactor is designed with the gold nanoparticle (NP)-modified carbon nanocage (Au@CNC) as a nanoreactor and meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a photosensitizer for cytochrome P450-mediated drug metabolism. By confining the cytochrome P450 3A4 (CYP3A4) enzyme and TCPP inside the pores of Au@CNC, a high metabolic activity is achieved by using 7-ethoxytrifluoromethyl coumarin as the substrate because of the three-dimensional hierarchical porous structure, large surface area, and fast electron transfer capacity of Au@CNC. It is noted that owing to the presence of AuNPs inside CNC, the surface hydrophilicity of CNC is much improved, which further promotes the catalytic activity of the CYP3A4 enzyme. To our knowledge, this is the first attempt to apply CNC as a bionanoreactor for NADPH-free and light-driven in vitro drug metabolism. In addition, the presented bionanoreactor exhibits a variety of advantages in terms of fast response, short assay time (10 min), high sensitivity, and good selectivity, which are expected to expedite drug screening and render potential advances in drug discovery and development.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

.Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7–25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.

Paper-based enzymatic platform coupled to screen printed graphene-modified electrode for the fast neonatal screening of phenylketonuria

IntroductionThe PKU is an inborn error of amino acid metabolism, in which phenylalanine (Phe) accumulated in the blood causing alterations at the central nervous system. We report a novel paper-based enzymatic platform coupled to screen printed graphene-modified electrode for the neonatal screening of phenylketonuria (PKU0. MethodsThe paper-based analytical device coupled to electrochemical detection (EPAD) is based on the use of paper microzones modified with phenylalanine dehydrogenase enzyme (PheDH). The modified PADs were placed on the surface of an electrode modified with electrochemically reduced graphene (ERGO). PheDH in the presence of NAD+ catalyzes the reversible deamination of Phe to form phenylpyruvate, ammonia, and NADH. The electrochemical oxidation of NADH was monitored by differential pulse amperometry (DPA) at 0.6 V. The method was linear in the concentration range from 1 to 600 μmol/L of Phe with a LOQ of 1 μmol/L and LOD of 0.2 μmol/L. Within day precision was 5.7% across 3 levels of control samples. Between-day precision was 8.3%. The comparison with the standard Phe enzyme assay kit showed good agreement. The time required for the overall assay was <5 min. The non-sophisticated equipment required, the short assay time and the appropriate LOQ and LOD achieved by our EPAD make it an attractive and easy to use alternative compared to existing methods applied to the screening of PKU in neonatal samples.

A novel strategy for rapid detection of bacteria in water by the combination of three-dimensional surface-enhanced Raman scattering (3D SERS) and laser induced breakdown spectroscopy (LIBS)

In this study, a novel strategy based on the combination of 3D SERS and LIBS was developed for qualitative and quantitative detection of bacteria. SERS-active Ag nanoparticles (AgNPs) were prepared by an improved in situ synthesis method, and reproducible SERS spectra of bacteria were obtained by natural evaporation of a droplet of the in situ synthesized sample. Four types of bacteria were classified via principal component analysis (PCA) and hierarchy cluster analysis (HCA). LIBS acted as a quantitative technique for bacterial detection based on the intracellular mineral cations, and bacteria could be detected in a linear range of 5 × 103–5 × 107 CFU mL−1, with recovery values ranging from 81.0% to 101.7%. The whole detection process including sample preparation and detection could be completed in approximately 30 min. With short assay time and simple operation, the proposed strategy has showed great potential for bacterial analysis.

Highly sensitive detection of high-risk bacterial pathogens using SERS-based lateral flow assay strips

Bacterial pathogens such as Yersinia pestis, Francisella tularensis, and Bacillus anthracis are classified into the highest rank of potential bioterrorism agents. Colorimetric lateral flow assay (LFA) strips are commercially available but these conventional strips have drawbacks in terms of low sensitivity and limit of quantitative analysis. Therefore, there is an urgent need for a new sensing platform to detect these pathogens in the early contamination stage. In this study, a novel surface-enhanced Raman scattering (SERS)-based LFA strip was developed for sensitive detection of bacterial pathogens. Target-specific SERS nanotags (Raman reporter-labeled gold nanoparticles) were used as an alternative to the gold nanoparticles in conventional LFA strips. Using these SERS nanotags the presence of bacteria could be identified through a simple color change in the test line. Additionally, highly sensitive and accurate quantitative analysis could be performed by monitoring the characteristic Raman peak intensity of SERS nanotags that were captured in the test line. This highly sensitive method required a short assay time (15 min) and a tiny volume of pathogen sample (40 μL). We believe that the proposed SERS-based LFA technique has great potential as a valuable tool in the early detection of specific bacterial pathogens in the field due to its excellent analytical sensitivity.

Comparative Study of DNA Circuit System-Based Proportional and Exponential Amplification Strategies for Enzyme-Free and Rapid Detection of miRNA at Room Temperature.

Because circulating microRNAs (miRNAs) have been recognized as a new class of blood-based biomarkers for various diseases, a significant challenge has been the development of point-of-care testing (POCT) systems based on detection of circulating miRNAs directly from serum. A promising approach to POCT systems is considered to be the development of enzyme-free and isothermal detection systems. Here, two types of DNA circuit system based on proportional and exponential amplification strategies were constructed using double-stranded DNA-modified magnetic beads (dsDNA-MBs) and their performances for detection of miRNA were studied comparatively. Both proportional and exponential amplification DNA circuit systems enabled the detection of target miRNA (miR-141) at room temperature without the need for additional enzymes because miR-141 acted as a catalyst for successive toehold-mediated DNA displacement reactions. A significant increase in the noise fluorescence signal was observed for the exponential amplification DNA circuit system because of the leakage (undesired DNA displacement reaction) revealed by the kinetic study on each DNA displacement reaction. Nevertheless, the exponential amplification DNA circuit system showed a lower limit of detection (LOD: 46 pM) and shorter assay time (15 min) compared to those of the proportional amplification DNA circuit system (LOD: 103 pM at 180 min). It is most likely that the exponential amplification DNA circuit system enabled amplification of both the signals and target miR-141, whereas the proportional amplification DNA circuit system enabled amplification of the signals alone. In addition, the exponential amplification DNA circuit system was able to discriminate between mismatched base sequences in miR-200 family members and specifically detect miR-141 even in the presence of serum. These findings are important for the rational design for POCT systems.

Magnetically controlled fluorescence aptasensor for simultaneous determination of ochratoxin A and aflatoxin B1

Development of an efficient method for the simultaneous detection of two highly concerning mycotoxins, ochratoxin A (OTA) and aflatoxin B1 (AFB1), is of great significance on food safety monitoring. Herein, a magnetically controlled fluorescence aptasensor for simultaneous determination of OTA and AFB1 has been successfully developed. The working principle of the aptasensor is based on the specific aptamer-mycotoxin recognition and further leads to the partial release of two distinguishable fluorescence labels from the magnetic carriers. Through the magnetic separation, the reporter probes in the supernatant solution can be collected and converted into a sensitive fluorescence signal with dual emission peaks. This aptasensor provided a wide detection range of 2 pg mL−1 − 5 ng mL−1 for OTA and 5 pg mL−1 − 10 ng mL−1 for AFB1. The new easy-to-wash and simple-to-use approach offers a simultaneous and high selective detection with high sensitivity (limits of detection of 0.67 and 1.70 pg mL−1 for OTA and AFB1, respectively). Remarkable accuracy (relative standard deviation < 5.6%) during the mycotoxins determination as well as excellent quantitative recoveries (95–108%) during the analysis of the spiked corn samples were also achieved. This simple aptasensing scheme provides a new avenue for high throughput screen of dual mycotoxins due to its simple manipulation, short assay times, high selectivity and sensitivity.

A Self-Assembly Fluorescence Sensing Platform for Glutathione Detection Based on Eco-Friendly Quantum Dots and MnO₂ Nanosheets.

Glutathione (GSH), a thiol-containing tripeptide, has a pivotal role in maintaining the normally physiological environment in biology. Here, we report a novel self-assembly fluorescence resonance energy transfer (FRET) sensing platform for GSH detection by using the environment-friendly Cu:Zn-In-S/ZnS quantum dots (QDs) and MnO2 nanosheets. In this assay, fluorescence of Cu:Zn- In-S/ZnS QDs could be quenched by MnO2 nanosheets based on FRET. Upon the addition of GSH, MnO2 nanosheets were reduced into Mn2+ ions because of the rapid redox reaction, and consequently the fluorescence of QDs was recovered. A good linear relationship was obtained from 1 to 20 μM for the detection of GSH with a low detection limit of 500 nM. Meanwhile, the sensing system also exhibited high selectivity against other electrolytes and biomolecules. Moreover, the satisfactory results in the determination of GSH in serum samples indicate that the assay holds great potential for detecting GSH in biological fluids. This method shows many advantages including high sensitivity, short assay time, good simplicity as well as label-free and environmental friendly, which is expected to facilitate the GSH quantification relating to biological and biomedical studies.

Combined use of vancomycin-modified Ag-coated magnetic nanoparticles and secondary enhanced nanoparticles for rapid surface-enhanced Raman scattering detection of bacteria.

BackgroundPathogenic bacteria have always been a significant threat to human health. The detection of pathogens needs to be rapid, accurate, and convenient.MethodsWe present a sensitive surface-enhanced Raman scattering (SERS) biosensor based on the combination of vancomycin-modified Ag-coated magnetic nanoparticles (Fe3O4@Ag-Van MNPs) and Au@Ag nanoparticles (NPs) that can effectively capture and discriminate bacterial pathogens from solution. The high-performance Fe3O4@Ag MNPs were modified with vancomycin and used as bacteria capturer for magnetic separation and enrichment. The modified MNPS were found to exhibit strong affinity with a broad range of Gram-positive and Gram-negative bacteria. After separating and rinsing bacteria, Fe3O4@Ag-Van MNPs and Au@Ag NPs were synergistically used to construct a very large number of hot spots on bacteria cells, leading to ultrasensitive SERS detection.ResultsThe dominant merits of our dual enhanced strategy included high bacterial-capture efficiency (>65%) within a wide pH range (pH 3.0–11.0), a short assay time (<30 min), and a low detection limit (5×102 cells/mL). Moreover, the spiked tests show that this method is still valid in milk and blood samples. Owing to these capabilities, the combined system enabled the sensitive and specific discrimination of different pathogens in complex solution, as verified by its detection of Gram-positive bacterium Escherichia coli, Gram-positive bacterium Staphylococcus aureus, and methicillin-resistant S. aureus.ConclusionThis method has great potential for field applications in food safety, environmental monitoring, and infectious disease diagnosis.

A facile fluorescence lateral flow biosensor for glutathione detection based on quantum dots-MnO2 nanocomposites

A newly designed quantum dots and manganese dioxide nanosheets nanocomposite-based fluorescence lateral flow biosensor was developed for rapid detection of glutathione (GSH). In this biosensing system, nanocomposites of biotinylated quantum dots (Biotin-QDs) and MnO2 nanosheets were first fixed on the conjugation pad of the test strip. The addition of GSH could trigger the decomposition of nanocomposites by digesting MnO2 nanosheets and consequently release Biotin-QDs. Under the capillary effect, the free Biotin-QDs would continue to move forward and be captured by streptavidin (SA) on the test line due to the specific binding between biotin and SA. Thereafter, an obvious fluorescent enhancement can be observed on the test line with a portable strip reader as well as naked eyes under irradiation by a 365 nm handheld ultraviolet lamps, which was used successfully for the quantitative detection of GSH with a short assay time of 20 min. Furthermore, this biosensor was utilized for GSH detection in diluted HeLa cells extracts (1%) samples with satisfactory results, which can meet low concentration detection for GSH with a good linear relationship in the range from 0.05 to 1.2 mM. Holding the advantages such as simple, convenient, rapid and cost effective, this developed fluorescence lateral flow biosensor has a great potential to be used for GSH detection in point of care diagnosis.

Citrate-capped silver nanoparticles as a probe for sensitive and selective colorimetric and spectrophotometric sensing of creatinine in human urine

Urinary creatinine concentration is a critical physiological parameter that enables reliable assessment of patient renal function and diagnosis of a broad spectrum of diseases. In this study, a simple and inexpensive sensor comprising monodisperse, citrate-capped silver nanoparticles (cc-AgNPs) was developed, which enabled rapid, sensitive and selective quantitation of creatinine directly in unprocessed urine. The mechanism of this sensor entails the creatinine-mediated aggregation of the cc-AgNPs (within 1 min) under alkaline conditions (pH 12). This is attributed to the tautomerization of creatinine to its amino anionic species at alkaline pH, which cross-link the cc-AgNPs via hydrogen bond networks with the negatively charged citrate caps. Creatinine elicited visibly-discernable color changes of the cc-AgNPs colloids in a concentration-dependent manner up to 10 μM. UV-visible spectroscopic analyses of the cc-AgNPs revealed that creatinine elicited a concentration-dependent decrease in intensity of the localized surface plasmon resonance (LSPR) band centered around 403 nm, with a concomitant increase in intensity of the red-shifted LSPR band at 670 nm. This observation denotes a creatinine-mediated increase in cc-AgNP particle size via aggregation, as confirmed by transmission electron microscopy analysis. The cc-AgNP sensor exhibited a linear correlation between the A670/A403 extinction ratio and creatinine concentration range of 0–4.2 μM in aqueous solutions (R2 = 0.996), and a low detection limit of 53.4 nM. Hence, the simplicity, short assay time, and high sensitivity and selectivity of our cc-AgNP sensor affirms its utility as a creatinine monitoring assay for low-resource, point-of-care settings.

Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for DNA Methyltransferase Activity and Inhibition.

DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes. However, conventional assays proposed for DNA methyltransferase activity are laborious and discontinuous. We have proposed a novel method for real-time monitoring of the activity and kinetics of Escherichia coli DNA adenine methyltransferase (Dam) using nanopore technique coupled with enzyme-linkage reactions. A double-stranded DNA probe AB having a recognition sequence 5'-GATC-3' for both Dam and MboI restriction endonuclease was prepared. Dam catalyzed the methylation of substrate probe AB, which blocked the cleavage reaction of MboI, while the absence of Dam resulted in cleavage of nonmethylated probe AB into four ssDNA fragments by MboI. When tested with nanopore, double-stranded methylated probe AB generated long-lived events, distinguished clearly from MboI-cleavage-mediated ssDNA fragments that generated only spikelike events. The proposed method has a detection limit of 0.03 U/mL for Dam in a short assay time of about 150 min. This sensing system is easy to perform, simple to design and circumvents the use of radioactive substances, resulting in efficient detection of the activity of Dam even in complex matrixes like human serum sample. Furthermore, it has the potential to screen Dam-targeted inhibitor drugs which may assist in the discovery of new anticancer medicines. This method is general and could be extended easily for monitoring activity of a wide variety of methyltransferases by coupling with their corresponding methylation-sensitive endonucleases.

A novel non-destructive manner for quantitative determination of plumpness of live Eriocheir sinensis using low-field nuclear magnetic resonance

The present study investigated the quantitative and non-destructive determination of Eriocheir sinensis' plumpness during four mature stages using low field-nuclear magnetic resonance (LF-1H NMR). Normalized lipid volume of live E. sinensis was calculated from Sept to Dec using 3D LF-1H nuclear magnetic imaging (MRI) and the validity of proposed technique was compared and verified with traditional Soxhlet extraction and live dissection method, respectively. The results showed the plumpness of female E. sinensis was higher than that of male ones from Sept to Dec and the highest plumpness of male and female E. sinensis reached 99,436.44 and 109,207.15mm3 in Oct. The normalized lipid volume of live male and female E. sinensis had a positive correlation with lipid content. This proposed method with short assay time, favorable selectivity, and accuracy demonstrated its application potential in grading regulation and quality evaluation of live E. sinensis.

A novel colorimetric strategy for sensitive and rapid sensing of ascorbic acid using cobalt oxyhydroxide nanoflakes and 3,3′,5,5′-tetramethylbenzidine

In this work, cobalt oxyhydroxide nanoflakes (CoOOH nanoflakes) were reported to possess a high oxidizability toward TMB for the first time and based on which we have developed a new strategy for qualitative and quantitative detection of ascorbic acid (AA). In our design, CoOOH nanoflakes can directly oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to oxTMB with an obvious blue color emerging, and AA can selectively inhibit the oxidation of TMB to oxTMB by resolving CoOOH nanoflakes to Co2+, resulting in a decrease in the absorbance of oxTMB and bleaching of blue color. As such, the concentration of AA could be identified very easily by UV–vis spectroscopy and bare eyes. Under optimal conditions, this sensing platform showed a highly sensitive response to AA with a detection limit of 142nM and a short assay time of 5min. This assay was also successfully used to quantify AA in human serum samples and practical food samples. Importantly, using this fabricated sensing strategy, we have further developed a paper-based strip sensor for AA detection with satisfactory results, showing its great potential for point of care applications.