Solid-phase recombinase polymerase amplification using an extremely low concentration of a solution primer for sensitive electrochemical detection of hepatitis B viral DNA.

Recombinase polymerase amplification (RPA) is considered one of the best amplification methods for realizing a miniaturized diagnostic instrument; however, it is notably challenging to obtain low detection limits in solid-phase RPA. To overcome these difficulties, we combined solid-phase RPA with electrochemical detection and used a new concentration combination of three primers (surface-bound forward primer, solution reverse primer, and an extremely low concentration of solution forward primer). When solid-phase RPA was performed on an indium tin oxide (ITO) electrode modified with a surface-bound forward primer in a solution containing a biotin-terminated solution reverse primer, an extremely low concentration of a solution forward primer, and a template DNA or genomic DNA for a target gene of hepatitis B virus (HBV), amplification occurred mainly in solution until all the solution forward primers were consumed. Subsequently, DNA amplicons produced in solution participated in solid-phase amplification involving surface-bound forward primer and solution reverse primer. Afterward, neutravidin-conjugated DT-diaphorase (DT-D) was attached to a biotin-terminated DNA amplicon on the ITO electrode. Finally, chronocoulometric charges were measured using electrochemical-enzymatic redox cycling involving the ITO electrode, 1,4-naphthoquinone, DT-D, and reduced ß-nicotinamide adenine dinucleotide. The detection limit for HBV was measured using microfabricated electrodes and was found to be approximately 0.1 fM. This proposed method demonstrated better amplification efficiency for HBV genomic DNA than solid-phase RPA without using additional solution primer and asymmetric solid-phase RPA.

Washing-Free Electrochemical Detection of Amplified Double-Stranded DNAs Using a Zinc Finger Protein.

Recombinase polymerase amplification (RPA) has been combined with electrochemical detection for simple and rapid point-of-care testing. However, there are two major hindrances to this simple and rapid testing: (i) washing or purification steps are required to remove unbound labeled probes and interfering species in the sample; (ii) it is difficult to quantify double-stranded DNA (dsDNA) electrochemically by using biospecific affinity binding without dsDNA denaturation. In the present study, we describe a wash-free and rapid electrochemical method to detect RPA-amplified dsDNAs using a zinc finger protein, Zif268. Electrochemical detection is achieved using proximity-dependent electron mediation of ferrocenemethanol between a glucose-oxidase (GOx) label and an electrode, which differentiates the specifically electrode-bound and -unbound labels without a washing or purification step. RPA-amplified dsDNA containing a biotin-terminated forward primer is specifically bound to a neutravidin-modified electrode, and GOx-conjugated Zif268 is specifically bound to the dsDNA. The whole detection is performed within 17 min (15 min for the RPA reaction and <2 min for the electrochemical measurement). Electrochemical detection is carried out without an additional incubation period, because the specific binding between Zif268 and the dsDNA occurs during the RPA reaction. The detection method could discriminate between target template DNA of Piscirickettsia salmonis and nontarget DNAs (random sequence and calf thymus DNA). The detection limit for the target DNA is approximately 300 copies in 13.2 µL, indicating that the detection method is ultrasensitive. We believe that the method could offer a promising solution for simple and rapid point-of-care testing.

Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids.

Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

An ultrasensitive and incubation-free electrochemical immunosensor using a gold-nanocatalyst label mediating outer-sphere-reaction-philic and inner-sphere-reaction-philic species.

This communication reports a new nanocatalytic scheme based on the facts that the redox reaction between a highly outer-sphere-reaction-philic (OSR-philic) species and a highly inner-sphere-reaction-philic (ISR-philic) species is slow and that an OSR- and ISR-philic Au-nanocatalyst label can mediate the two different types of redox species. This scheme allows highly sensitive and incubation free detection of creatine kinase-MB.