Simple and Sensitive Quantification of MicroRNAs via PS@Au Microspheres-Based DNA Probes and DSN-Assisted Signal Amplification Platform

Identifying the microRNA (miRNA) expression level can provide critical information for early diagnosis of cancers or monitoring the cancer therapeutic efficacy. This paper focused on a kind of gold-nanoparticle-coated polystyrene microbeads (PS@Au microspheres)-based DNA probe as miRNA capture and duplex-specific nuclease (DSN) signal amplification platform based on an RGB value readout for detection of miRNAs. In virtue of the outstanding selectivity and simple experimental operation, 5′-fluorochrome-labeled molecular beacons (MBs) were immobilized on PS@Au microspheres via their 3′-thiol, in the wake of the fluorescence quenching by nanoparticle surface energy transfer (NSET). Target miRNAs were captured by the PS@Au microspheres-based DNA probe through DNA/RNA hybridization. DSN enzyme subsequently selectively cleaved the DNA to recycle the target miRNA and release of fluorophores, thereby triggering the signal amplification with more free fluorophores. The RGB value measurement enabled a detection limit of 50 fM, almost 4 orders of magnitude lower than PS@Au microspheres-based DNA probe detection without DSN. Meanwhile, by different encoding of dyes, miRNA-21 and miRNA-10b were simultaneously detected in the same sample. Considering the ability for quantitation, high sensitivity, and convenient merits, the PS@Au microspheres-based DNA probe and DSN signal amplification platform supplied valuable information for early diagnosis of cancers

Effective Bioactivity Retention of Low-Concentration Antibodies on HFBI-Modified Fluorescence ICTS for Sensitive and Rapid Detection of PSA

Nowadays, increasing analytical sensitivity is still a big challenge in constructing membrane-based fluorescence immunochromatography test strips (FICTS). However, the bioactivity of antibody (Ab) immobilized on the test line (T line) of porous nitrocellulose membrane (PNM), which directly influences the analytical sensitivity, is less studied. In this work, a novel amphiphilic hydrophobin (HFBI) protein was introduced to modify the T line to effectively retain the Abs’ bioactivity. The results indicated that HFBI could self-assemble on the PNM and immobilize the Abs in the “stand-up” orientation. Compared with the conventional FICTS, the HFBI-modified FICTS with only 0.2 mg/mL of monoclonal Abs on T line enable more accurate quantitative detection and better sensitivity (0.06 ng/mL for prostate specific antigen), which is more than 2 orders of magnitude lower than that of the conventional FICTS with the same concentration of monoclonal Abs on T line. Furthermore, the accuracy of this HFBI-modified FICTS was investigated by testing 150 clinical serum samples and the detection results were coincident with those by electrochemiluminescence immunoassay. Our results provide a novel and promising strategy of Ab immobilization on FICTS for near-patient and point-of-care application

Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence “Turn-on” with Enzyme–Antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg

At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence “turn-on” signals. In this system, detection antibodies (Ab₂) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine₁₁₀ with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10^–8 ng mL^–1 and 5 × 10^–4 IU mL^–1, respectively, which were at least 10⁴ times lower than those of the conventional fluorescence assay and 10⁶ times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications