Ultrasensitive Biosensors Detecting m6A in Blood: Achieving Early Screening and Typing of Tumors.

N6-methyladenosine (m6A) modification is one of the most widespread RNA modifications in eukaryotes and is involved in cancer development and progression by regulating oncogene expression. Herein, a reticulated rolling circle amplification (RCA) cascade reaction was used to construct a novel electrochemical biosensor for ultrasensitive detection of m6A, employing ferrocene-tyramine (Fc-Tyr) molecules as electroactive probes. In this strategy, the RCA cascade reaction not only amplifies specific circular DNA in the designed template to reduce the binding with similar nucleic acid sequences but also generates a long ssDNA through multiple repetitions to capture a large number of electrochemical signal probes and achieve the amplification of electrochemical biosensing signals. The developed biosensor demonstrated high selectivity and sensitivity toward m6A in the range of 0.5 pM-150 nM, with a detection limit of 14.07 fM. Meanwhile, total RNA extracted from cell samples was analyzed for m6A expression levels using the developed biosensor and a commercial colorimetric immunoassay, the biosensor and immunoassay showed consistent results. In addition, m6A levels in clinical serum samples were assessed using the developed electrochemical biosensor, which showed that m6A expression was much lower in healthy individuals than in cancer patients, therefore the biosensor is promising for cancer typing. This study provides a new method for rapid and convenient tumor marker detection in clinical practice, as well as a new idea for sensitive detection of other biomolecules.

An ultrasensitive electrochemical biosensor with dual-amplification mode and enzyme-deposited silver for detection of miR-205-5p.

An electrochemical biosensor based on dual-amplified nucleic acid mode and biocatalytic silver deposition was constructed using catalytic hairpin assembly-hybrid chain reaction (CHA-HCR). The electrochemical detection of silver on the electrode by linear sweep voltammetry (LSV) can be utilized to quantitatively measure miR-205-5p since the amount of silver deposited on the electrode is proportional to the target nucleic acid. The current response values exhibit strong linearity with the logarithm of miR-205-5p concentrations ranging from 0.1 pM to 10 µM, and the detection limit is 28 fM. A consistent trend was found in the results of the qRT-PCR and electrochemical biosensor techniques, which were employed to determine the total RNA recovered from cells, respectively. Moreover, the constructed sensor was used to assess miR-205-5p on various cell counts, and the outcomes demonstrated the excellent analytical efficiency of the proposed strategy. The recoveries ranged from 97.85% to 115.3% with RSDs of 2.251% to 4.869% in human serum samples. Our electrochemical biosensor for miR-205-5p detection exhibits good specificity, high sensitivity, repeatability, and stability. It is a potentially useful sensing platform for tumor diagnosis and tumor type identification in clinical settings.

Detection of methyltransferase activity and inhibitor screening based on rGO-mediated silver enhancement signal amplification strategy.

DNA methylation refers to the chemical modification process of obtaining a methyl group by the covalent bonding of a specific base in DNA sequence with S-adenosyl methionine (SAM) as a methyl donor under the catalysis of methyltransferase (MTase), which is related to the occurrence of multiple diseases. Therefore, the detection of MTase activity is of great significance for disease diagnosis and drug screening. Because reduced graphene oxide (rGO) has a unique planar structure and remarkable catalytic performance, it is not clear whether rGO can rapidly catalyze silver deposition as an effective way of signal amplification. However, in this study, we were pleasantly surprised to find that using H2O2 as a reducing agent, rGO can rapidly catalyze silver deposition, and its catalytic efficiency of silver deposition is significantly better than that of GO. Therefore, based on further verifying the mechanism of catalytic properties of rGO, we constructed a novel electrochemical biosensor (rGO/silver biosensor) for the detection of dam MTase activity, which has high selectivity and sensitivity to MTase in the range of 0.1 U/mL to 10.0 U/mL, and the detection limit is as low as 0.07 U/mL. Besides, this study also used Gentamicin and 5-Fluorouracil as inhibitor models, confirming that the biosensor has a good application prospect in the high-throughput screening of dam MTase inhibitors.

An electrochemical immunosensor for the detection of Glypican-3 based on enzymatic ferrocene-tyramine deposition reaction.

An ultrasensitive electrochemical immunosensor based on signal amplification of the deposition of the electroactive ferrocene-tyramine (Fc-Tyr) molecule, catalyzed by horseradish peroxidase (HRP), was constructed for the detection of the liver cancer marker Glypican-3 (GPC3). Functional electroactive molecule Fc-Tyr is reported to exhibit both the enzymatic cascade catalytic activity of tyramine signal amplification (TSA) and the excellent redox properties of ferrocene. In terms of design, the low matrix effects inherent in using the magnetic bead platforms, a quasi-homogeneous system, allowed capturing the target protein GPC3 without sample pretreatment, and loading HRP to trigger the TSA, which induced a large amount of Fc-Tyr deposited on the electrode surface layer by layer as a signal probe for the detection of GPC3. The concept of Fc-Tyr as an electroactive label was validated, GPC3 biosensor exhibited high selectivity and sensitivity to GPC3 in the range of 0.1 ng mL-1-1 µg mL-1. Finally, the sensor was used simultaneously with ELISA to assess GPC3 levels in the serum of clinical liver cancer patients, and the results showed consistency, with a recovery of 98.33-105.35% and a relative standard deviation (RSD) of 4.38-8.18%, providing a theoretical basis for achieving portable, rapid and point of care testing (POCT) of tumor markers.