Reliable Digital Single Molecule Electrochemistry for Ultrasensitive Alkaline Phosphatase Detection

Abstract

Single molecule electrochemistry (SME) has gained much progress in fundamental studies, but it is difficult to use in practice due to its less reliability. We have solved the reliability of single molecule electrochemical detection by integration of digital analysis with efficient signal amplification of enzyme-induced metallization (EIM) together with high-throughput parallelism of microelectrode array (MA), establishing a digital single molecule electrochemical detection method (dSMED). Our dSMED has been successfully used for alkaline phosphatase (ALP) detection in the complex sample of liver cancer cells. Compared to direct measurement of the oxidation current of enzyme products, EIM can enhance signals by about 100 times, achieving signal-to-background ratio high enough for single molecule detection. The integration of digital analysis with SME can further decrease the detection limit of ALP to 1 aM relative to original 50 aM, enabling dSMED to be sensitively, specifically and reliably applied in liver cancer cells. The presented dSMED is enormously promising in exploring physical and chemical properties of single molecules, single biomolecular detection, or single-cell analysis.

Alkaline Phosphatase‐based Electrochemical Analysis for Point‐of‐Care Testing

“…With incorporation of recognition components for biomolecules, the devices can be applied to POCT. In 2016, the Zhang group developed a digital signal molecule electrochemical detection (dSMED) method as an ultrasensitive analytical method of ALP [23]. The devices contained the ALP molecule in microwells that convert PAPP to silver deposition using PAP as a reducing agent at each microwell.…”

Overview of Optical and Electrochemical Alkaline Phosphatase (ALP) Biosensors: Recent Approaches in Cells Culture Techniques

“…The estimated time to attain a steady-state was short and this shows that the electrochemical image with 256 pixels was achieved in 10.3 s using the LRC-device. A study by Wu et al (2016) developed a new digital single molecule electrochemical detection method (dSMED) strategy to address the problem of single molecule electrochemistry (SME) reliability based on the integration with the digital analysis in enzyme-induced metallization (EIM), which improves the application of SME in a biological system [165]. The created EIM can enhance electrochemical signals up to 100 times when compared to conventional techniques of direct oxidation of enzyme materials that offer better signal detection for single molecule detection.…”

Biosensor for Rapid and Sensitive Detection of Influenza Virus

“…For this purpose, the digital ELISA was integrated by digital analysis, bifunctional fluorescence magnetic nanospheres (bi-FMNs), and monolayer AuNPs-modified microelectrode array (MA). The digital analysis enables the virus to be detected at low concentration signals of "0" or "1", regardless of the concrete strength of signals [76,77]. The bi-FMNs were fabricated by co-immobilizing polyclonal anti-H7N9 HA antibody and alkaline phosphatase.…”