Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP).

Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as ∼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications.

A multiplex paper-based nanobiocatalytic system for simultaneous determination of glucose and uric acid in whole blood.

In this work, a versatile point-of-care assay platform based on a microfluidic paper-based analytic device (µPAD) was developed for the simultaneous detection of multiple targets. The µPAD with a central zone and six test zones is fabricated by a simple and inexpensive wax printing method. A flower-like hybrid nanocomplex synthesized with specific dual enzymes and Cu3(PO4)2 inorganic nanocrystals is spotted in the test zones on the µPAD, followed by the introduction of assay targets. Using dual-enzyme inorganic hybrid nanoflowers in the µPAD as nanobiocatalysts, which preserve the activity and enhance the stability of the enzymes, based on the H2O2-mediated catalytic oxidizing chromogenic reaction produced by glucose/uric acid, the developed multiplex paper-based nanobiocatalytic system is demonstrated to enable simultaneous and sensitive detection of glucose and uric acid with a detection limit of 60 and 25 µM, respectively. More importantly, it has been successfully used for detecting glucose and uric acid levels in human whole blood samples. The developed multiplex paper-based nanobiocatalytic system features very easy fabrication and operation, low cost, and high sensitivity and has promising prospects for a clinical multianalyte point-of-care test.