Search

Abstract: A microfluidic flow cell with embedded gold interdigitated array microelectrode (IDAM) was developed and integrated with magnetic nanoparticle-antibody conjugates (MNAC) into an impedance biosensor to rapidly detect pathogenic bacteria in ground beef samples. The flow cell consisting of a detection microchamber and inlet and outlet microchannels was fabricated by bonding an IDAM chip to a poly(dimethylsiloxane) (PDMS) microchannel. The detection microchamber with a dimension of 6mm×0.5mm×0.02mm and a volume of 60nL was used to collect bacterial cells in the active layer above the microelectrode for sensitive impedance change. MNAC were prepared by conjugating streptavidin-coated magnetic nanoparticles with biotin-labeled polyclonal goat anti-E. coli antibodies and were used in the separation and concentration of target bacteria. The cells of E. coli O157:H7 inoculated in a food sample were first captured by the MNAC, separated, and concentrated by applying a magnetic field, washed, and then suspended in mannitol solution and finally injected through the microfluidic flow cell for impedance measurement. This impedance biosensor was able to detect as low as 1.6×102 and 1.2×103 cells of E. coli O157:H7 cells present in pure culture and ground beef sample, respectively. The total detection time from sampling to measurement was 35min. Equivalent circuit analysis indicated that the bulk medium resistance, double layer capacitance, and dielectric capacitance were responsible for the impedance change due to the presence of E. coli O157:H7 cells on the surface of IDAM. Sample pre-enrichment, secondary antibodies on the microelectrode surface, and redox probes were not required in this impedance biosensor. [Copyright &y& Elsevier]

Abstract: In this study, a novel one-step label-free immunoassay using gold nanorods (GNRs) for sensitive detection of alpha-fetoprotein (AFP), one of the major markers for hepatocellular tumors, was successfully developed. GNRs were fabricated through seed-mediated growth procedure, and their surface was activated by complete replacement with alkanethiols for the attachment of antibodies to obtain GNR-antibody conjugates with special space conformation. Dramatic aggregation of GNRs was induced by the interaction between target proteins and capture antibodies, and confirmed by transmission electron microscopy (TEM) and Ultraviolet–visible (UV–vis) analysis. Associated wavelength red-shifts and intensity decreases in the absorption peak of GNRs were observed with increasing concentrations of target AFP. Therein the absorption intensity as sensing response showed excellent linearity with the concentration of AFP in logarithmic scale. It was illustrated that this method can be used reliably to detect AFP protein in phosphate-buffered saline (PBS) buffer at concentrations of picomolar level with a wide quantification range from 0.25 to 4.0nM. The complication of interfering proteins in the sample showed negligible effects on the detection results. Furthermore, the simple procedure, cost-effective and label-free features of this method offer advantages over common immunoassays. It is also expected that this new method would be applicable for detection of other tumor markers at low concentrations. [Copyright &y& Elsevier]

Point-of-care testing (POCT) of pathogens is important for avoiding food poisoning. Here, dual-functional manganese dioxide nanoclusters (MnO 2 NCs) were elaboratively used as the fluidic driver and signal amplifier for exploring a new power-free biosensor for POCT of Salmonella. First, H 2 O 2 in three chambers of a microfluidic chip was finger driven simultaneously to dissolve MnO 2 NCs, resulting in the production of O 2 to respectively push preloaded immune magnetic nanobeads (IMNBs), bacterial sample and immune MnO 2 NCs (IMONCs) into a sinusoidal convergence-divergence micromixer. After they were efficiently mixed and incubated in a serpentine channel, IMNB-bacteria-IMONC conjugates were obtained and magnetically separated in a multifunctional chamber. Then, two washing solution were successively driven to rinse these conjugates. Finally, colorless H 2 O 2 -TMB substrate was driven to the multifunctional chamber and catalyzed by the IMONCs on the conjugates into blue catalysate, which was analyzed by developed smartphone APP to determinate bacteria amount. The biosensor enabled quantitative detection of 63 CFU/mL Salmonella within 30 min. The complete bacteria detection procedure was achieved on a single microfluidic chip without any peripheral equipment, and it has great potential for POCT of bacteria. [Display omitted] • Dual-functional MnO 2 nanoclusters were elaborately used as fluidic driver and signal amplifier. • The whole bacterial detection procedure was implemented free-of-power in a microfluidic chip. • The sinusoidal convergence-divergence micromixer was verified with good mixing effect. • Smartphone App was self-developed to accurately measure color change of catalysate. • This microfluidic biosensor was able to detect Salmonella as low as 63 CFU/mL in 30 min. [ABSTRACT FROM AUTHOR]

A novel and simple colorimetric biosensor was developed by combining the unique optical properties of unmodified gold nanorods (AuNRs) with the amplification strategy of hybridization chain reaction (HCR), which was capable of detecting DNA sensitively and selectively. The detection mechanism is based on the dispersion/aggregation of AuNRs under the high concentration of salt. When target DNA introduces, the hybridization of hairpin DNA is triggered and a nicked double-helix DNA as HCR product forms which sticks tightly with AuNRs through a strong electrostatic adsorption and protects AuNRs from aggregation in high salt concentration condition. When no target DNA appears, AuNRs undergo aggregation due to the weak protection of hairpin DNA. The approach is able to detect target DNA in a range of 0–60 nM with a detection limit of 1.47 nM and exhibits high selectivity to distinguish fully matched and single-base mismatched DNA. Results demonstrate this method is label-free, modification-free, and enzyme-free, which holds great promise for routine sensing applications. [ABSTRACT FROM AUTHOR]

Acrylamide (AA) is a small molecule with neurotoxicity, potential carcinogen, and genotoxicity. Aptamers, known as “chemical antibodies”, are single-stranded DNA or RNA specific binding to their targets. Herein, aptamers specific to AA were first selected by a quartz crystal microbalance (QCM) combined with systematic evolution of ligands by exponential enrichment (SELEX) method. The selection started with a single-stranded DNA (ssDNA) library of 10 15 molecules randomized at central 45 nt. In each round, the binding affinity of ssDNA pool to AA was in real-time characterized by monitoring the change of frequency (Δ f ) before and after incubation with AA. After 14 rounds of positive selection and 4 rounds of counter selection, AA aptamer candidates were enriched, of which two aptamer candidates (A5 and C14) were selected and characterized by QCM and dot blot assay. The dissociation constants of A5 and C14 were 17.2 nmol L −1 and 115.2 nmol L −1 , respectively. Both A5 and C14 didn’t show affinity to acrylamide analogues, including acrylic acid, methacrylic acid, and methacrylamide. To enable rapid and specific detection of AA with the selected aptamers, a label-free colorimetric aptasensor was developed. The detection linear range of the aptasensor was from 0.5 to 10 μmol L −1 with a detection limit of 0.390 μmol L −1 . [ABSTRACT FROM AUTHOR]

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases. • A novel linear displacement isothermal amplification (LDIA) method was developed by using conventional primers. • Using strand displacement (SD) probes, an LDIA-SD method was established to realize probcleic acid-free extraction step and a pocket-sized device were used to realize its POCT applicate-based specific detection. • A nucleic acid-free extraction step and a pocket-sized device were used to realize its POCT applications. [ABSTRACT FROM AUTHOR]

In this study, a cost-effective nanowell structure was fabricated and utilized for the development of a nanowell-based quartz crystal microbalance (QCM) aptasensor for rapid, sensitive, and label-free detection of H5N1 avian influenza virus (AIV). A nanoporous gold film with a thickness of 120 nm and a pore size of ∼20 nm was prepared using a metallic corrosion method. Then, the nanoporous gold film was immobilized onto a gold electrode surface using a self-assembled monolayer to form a nanowell-based electrode. A specific H5N1 AIV ssDNA aptamer with a NH 2 conjugated 5′-terminal was used in the fabrication of the QCM aptasensor through covalent bonding. The stepwise assembly of the aptasensor was characterized by means of QCM. The result showed that the binding of target AIV H5N1 onto the immobilized aptamers decreased the sensor’s resonant frequency, and the frequency change correlated to the virus titer. We demonstrated that the developed nanowell-based QCM aptasensor could dramatically reduce detection time down to 10 min using a label-free assay. The detection range of 2 −4 to 2 4 hemagglutination units (HAUs)/50 μl was obtained with a detection limit of 2 −4 HAU/50 μl for AIV H5N1. The binding of target H5N1 virus onto the nanowell-based electrode surface was further confirmed by scanning electron microscopy (SEM). No interference was observed from non-target AIV subtypes of H1N1, H2N2, H7N2 and H5N3. The aptasensor using H5N1 aptamer was validated for the detection of AIV H5N1 in chicken tracheal swab samples. The developed aptasensor could be adopted for detection of other viruses. [ABSTRACT FROM AUTHOR]

A target-responsive hydrogel based fluorescent aptasensor was developed for rapid detection of avian influenza virus (AIV) H5N1. A specific aptamer against H5N1 was selected and two single-stranded DNAs (ssDNA 1 and ssDNA 2 ) were designed to be partially complementary to two ends of the aptamer. Both aptamer and ssDNA 1 were functionalized with acrydite at 5’-terminal for hydrogel synthesis by polymerization. Quantum dots (QDs), used as fluorescence reporters, were labeled at 5’-terminal of ssDNA 2 and quenchers for QDs were conjugated at 3’-terminal of the aptamer. Hybridization between aptamer and ssDNAs formed the crosslinker to complete the QD-aptamer hydrogel. Initially, without targets, the prepared hydrogel remained in a shrunken state because of the crosslinking in the polymer network and the QD was quenched. With target AIVs, the crosslinking was dissociated due to the binding reaction between the aptamer and target, resulting in the abrupt swelling of the hydrogel and the release of aptamer-quencher and ssDNA 2 -QD. The response of aptamer upon target binding was demonstrated by quartz crystal microbalance (QCM) method and the microstructure of hydrogel was characterized by SEM. Spectrophotometric results indicated fluorescent changes at the emission peak were correlated to the titers of H5N1. The total detection time from sampling to results was 30 min. The lowest detection limit was 0.4HAU with a detecting range of 2 −1.2 to 2 6 HAU 20 μL −1 . The size-dependent property of this aptasensor was examined quantitatively and qualitatively. This simple, low-cost, specific, and label-free aptasensor has great potential for the in-field rapid detection of AIV H5N1. [ABSTRACT FROM AUTHOR]

Salmonella Typhimurium (S. Typhimurium) is regarded as a major cause of foodborne diseases, which has been identified as a server threat to public health. Herein, an electrochemical sensing method was developed to detect S. Typhimurium in combination with polymerase chain reaction (PCR) and CRISPR/Cas12a (E-CRISPR biosensor). The target DNA extracted from the bacteria was amplified by PCR first, followed by the second amplification through the activation of collateral cleavage activity of Cas12a against signaling hairpin DNA probes, causing the release of electrochemical labels and a dramatic current decrease. The design of hairpin DNA on the electrode also reduced the steric hindrance effect for better Cas12a collateral cleavage efficiency, leading to improved detection performance. Under the optimized conditions, the proposed E-CRISPR biosensor allowed the sensitive detection of S. Typhimurium in a linear range from 6.7 × 101 to 6.7 × 105 CFU/mL with a limit of detection of 55 CFU/mL in pure culture. In addition, the E-CRISPR biosensor enables the detection of S. Typhimurium in spiked poultry meat with excellent accuracy and sensitivity. This CRISPR-based biosensor has the potential to provide an alternative way for the detection of foodborne pathogens in the food supply chain. • An E-CRISPR detection platform for Salmonella Typhimurium was developed. • The method could detect Salmonella Typhimurium down to 55 CFU/mL. • The applicability was verified for detection of Salmonella Typhimurium in poultry samples. [ABSTRACT FROM AUTHOR]

A flexible fluorescent sensing strategy for the recognition and detection of bisphenol A (BPA) has been proposed based on molecularly imprinted polymers (MIPs)-coated gold nanoclusters (AuNCs), by taking advantages of the high selectivity of MIPs and the strong fluorescence property of AuNCs. SiO 2 @AuNCs were initially prepared by making use of the powerful amido bonds between carboxyl-terminated AuNCs and amino-functionalized SiO 2 nanoparticles. Then MIPs-coated AuNCs were formed by anchoring MIP layer on the surface of SiO 2 @AuNCs via a sol–gel process. In the presence of imprinting template BPA, a Meisenheimer complex could be formed between BPA and the primary amino groups on the surface of the AuNCs, and the photoluminescent energy of AuNCs would be transferred to the complex, and thereby result in the fluorescence quenching of AuNCs. The fluorescence-quenching fractions of the sensor presented a satisfactory linearity with BPA concentrations over the range of 0–13.1 μM and the detection limit could reach 0.10 μM. Distinguished selectivity was also exhibited to BPA over other possibly competing molecules. Moreover, the sensor was successfully applied to determine BPA in seawater, and the average recoveries of BPA at three spiking levels ranged from 91.3 to 96.2% with relative standard deviations below 4.8%. This AuNCs-MIPs based sensor provided great potentials for recognition and determination of phenolic environmental estrogens in complicated samples. [ABSTRACT FROM AUTHOR]

• This was first time to use HAc to release curcumin as reporter. • ZnO NPs were used to cap MSNs to avoid premature release of curcumin. • Koch fractal mixer was developed and verified with excellent performance. • The biosensor was able to detect Salmonella as low as 101 CFU/mL in 1.5 h. • The mean recovery of the spiked chicken samples was ∼104 %. An acid-responsive microfluidic biosensor using curcumin (CUR) as signal reporter and ZnO-capped mesoporous silica nanoparticles (MSNs) for signal amplification was developed for sensitive and rapid detection of Salmonella in colorimetric and fluorescent modal. The MSNs were first incubated with CUR to obtain MSN@CUR nanoparticles (MC NPs). The MC NPs were then capped with ZnO nanoparticles to form MSN@CUR@ZnO nanoparticles (MCZ NPs) to prevent CUR from premature release, followed by modification with polyclonal antibodies (pAbs) against Salmonella Typhimurium (S. Typhimurium) to obtain immune MSN@CUR@ZnO@pAbs nanoparticles (MCZP NPs). The immune magnetic nanoparticles (MNPs), the S. Typhimurium cells and the MCZP NPs were conjugated in the microfluidic chip with a Koch fractal mixing channel to form MNP-bacteria-MCZP complexes. Finally, the acetic acid (HAc) was first time introduced to release CUR from the complexes, and both the color and fluorescence changes were measured to determine the concentration of Salmonella. This proposed biosensor was able to quantitatively detect S. Typhimurium ranging from 102 to 107 CFU/mL in 1.5 h and the lower detection limits were calculated to be 63 CFU/mL for colorimetric measurement and 40 CFU/mL for fluorescent measurement, respectively. The mean recovery was about 104 % for Salmonella in the spiked chicken samples. [ABSTRACT FROM AUTHOR]