Search

We developed an electrochemical impedance spectroscopy (EIS) biosensor based on bacteriophage long tail fibre proteins (LTFPs) to capture and detect Salmonella. First, based on the genetic information of a Salmonella enterica bacteriophage (SEP37), we found that the genes ORF99, ORF100, and ORF256 encode proteins associated with host recognition. We used the pETdue1 plasmid to construct ORF99–ORF100 and ORF256 co-expression vectors to express (LTFPs) in Escherichia coli and used purified LTFPs as specific biorecognition elements. Then, we fixed gold nanoparticles (AuNPs), cysteamine (Cys), and LTFPs on a gold disk electrode surface via layer-by-layer assembly to construct an EIS biosensor. Salmonella in the sample can be captured by LTFPs on the electrode surface and reduce the electron transfer sites. We applied the EIS biosensor to detect Salmonella spiked in lake water. It showed a good linear relationship within the concentration range of 10 ∼ 1 × 106 colony-forming units (CFU)/mL. The detection limit was 9 CFU/mL. This method based on bacteriophage LTFPs does not require complex pretreatment steps and extends the application of tail fibre proteins as a biorecognition element in the detection field. [Display omitted] • ORF99-100 and ORF256 were inserted into plasmid to construct co-expression vector. • The long tail fibre protein of phage SEP37 was co-expressed in E. coli. • This protein was used as bio-recognition element to construct an EIS biosensor. • This biosensor can quickly detect Salmonella in lake water samples. • This method allowed a detection limit of 9 CFU/mL for Salmonella. [ABSTRACT FROM AUTHOR]

Two main challenges for biosensors facing may be analytical sensitivity and selectivity. Ultra-highly specific protein molecularly imprinted polymers (MIPs) with characteristics of easy preparation, chemical stability, convenient assembly with transducer, offer unprecedented opportunities for high-performance recognition towards targets. A dual-responsive electrochemical biosensor was demonstrated, where integrated both artificial protein MIPs and natural hyaluronic acid (HA) probes into one flexible electrode to detect biomarker CD44. Their efficacy towards target of two types of probes were elucidated and contrasted. Dual-channel screen-printed electrodes (SPEs) were designed innovatively. As one responsive channel, protein MIPs were synthesized through a polymerization procedure between functional monomer of alginate gel, a biocompatible antifouling chemical, and template protein CD44. Natural HA probes were immobilized to produce the other responsive channel. Such binary channels realized highly sensitive and selective target quantifying with lower limit of detections (LODs) of 1.41 × 10−5 ng/mL for MIPs-based and 1.51 × 10−5 ng/mL for HA-based channels, attributing to superior specificity, antifouling property and biocompatibility of MIPs and HA, satisfying stability of SPEs, high sensitivity of electrochemical techniques. The dual-responsive strategy can be extended to other batched integration of functionalized materials and active biomolecules into electrochemical and electro-catalytic biosensors towards multiple components to monitor physical signs and diagnose human diseases. [Display omitted] • A dual-responsive biosensor based on artificial and natural probes was constructed. • A dual-channel screen-printed electrode was designed and manufactured innovatively. • Artificial protein imprinted probe was synthesized and contrasted with natural probe. • Binary channels achieved ultra-highly sensitive and selective detection towards CD44. • Binary channels realized target quantification in complex human serum. [ABSTRACT FROM AUTHOR]

Abstract: A highly sensitive integrated polarimetric interferometer biosensor with improved long-time stability and simple operation was prepared by using a novel prism-chamber assembly and an inexpensive waveguide made by sputtering a tapered nanometric layer of Ta2O5 on a single-mode glass waveguide. By comparing the measured refractive-index (RI) sensitivities with those simulated based on a four-layer homogeneous waveguide, both the equivalent thicknesses (T eq) for the tapered Ta2O5 layers and a severe dependence of RI sensitivity on T eq were obtained. Addition of 1g of water in 100g of a Chinese liquor (alcohol concentration=46% (v/v)) was easily detected by the sensor. Monitoring of anti-human IgG adsorption with a waveguide of T eq =31.99nm indicates that the antibody coverage required for inducing a phase-different change of Δϕ = π is less than 0.012 monolayer. The same waveguide presents a quasi-linear dependence of Δϕ on water temperature with the slope of d(Δϕ)/dT =−28.50°/°C to which the contribution by the thermo-optical effect of the waveguide is 4.24°/°C, equivalent to a liquid RI change of Δn c =1.41×10−5. The interferometer exhibits the promising potential for chemical and biological analyses because of its outstanding characteristics. [Copyright &y& Elsevier]

Melioidosis caused by Burkholderia pseudomallei (Bp) infection has high mortality in tropical and subtropical regions. A rapid and sensitive method for its detection is urgently needed. Herein, a ratiometric fluorescent sensor with dual recognition sites of target was designed for sensitive detection of Bp DNA. The sensor was constructed with a TAMRA-labeled DNA as probe, which could adsorb on polymer dots (Pdots) at acidic pH to produce TAMRA fluorescence at the excitation wavelength of Pdots via fluorescence resonance energy transfer (FRET). In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a was activated with the dual recognition sites, which digested the probe DNA to weaken the FRET. With the fluorescence ratio of Pdots to TAMRA as the detection signal, a ratiometric fluorescent biosensing was thus achieved. This method with dual CRISPR/Cas12a trans-cleavage assisted signal enhancement showed a detectable range of target DNA from 1.0 pM to 10 nM, and could be successfully used to detect Bp DNA extract with a detection time of 40 min. The excellent performance such as high sensitivity, good specificity, acceptable accuracy and short analytical time along with convenient operation demonstrated its potential in clinic diagnosis of melioidosis. • A dual CRISPR/Cas12a trans-cleavage assisted signal enhancement strategy is proposed for biosensing of biomarker. • The proposed biosensing method based on ratiometric fluorescence has been used for detection of Burkholderia pseudomallei. • The proposed method shows a wide detection range with LOD of 1 pM and a detection time of 40 min with simple operation. • This proposed strategy possesses excellent performance and good extendability for target DNA analysis. [ABSTRACT FROM AUTHOR]

Dengue caused by dengue virus (DENV) is a highly pathogenic viral disease. Early detection of pathogens is very important for patient treatment and pathogen control. This work designed a dual signal amplification strategy for sensitive fluorescent detection of DENV RNA. The dual signal amplification was achieved by using a target-triggered hybridization chain reaction (HCR) to connect multiplex proximity ligation activated CRISPR-Cas12a (pCRISPR-Cas12a). The HCR was triggered by the recognition of DENV RNA to a hairpin probe (H3), which led to a product to initiate the continuous hybridization of a couple of hairpins. Here one of the hairpins (H2) was designed to contain two domains (a and b), and domains a and b from two H2 formed a proximity ligation sequence (PLS) to connect the multiplex pCRISPR-Cas12a onto the HCR product, in which domain a activated the Cas12a, and domain b enhanced the enzymatic activity by about 5 times. Upon the enzymatic cleavage of single-stranded DNA labeled with a fluorophore and quencher at each end (ssDNA-FQ), a strong fluorescent signal was produced for DENV RNA analysis, which showed a linear range from 1 pM to 10 nM and a detection limit of 51 fM. The excellent performance of the proposed homogenous fluorescent assay with high sensitivity and superior accuracy endowed this strategy broad application prospects in clinical disease diagnosis. [Display omitted] • A universal fluorescence biosensing strategy was developed for highly sensitive detection of Dengue virus RNA. • The parallel connection of proximity sequence enhanced CRISPR-Cas12a was achieved through target-triggered HCR. • This proposed homogenous detection method showed a wide detection range and 50 fM-level detection limit. • This proposed strategy possesses good accuracy, high precision, and good extendability for target analysis. [ABSTRACT FROM AUTHOR]