Combining magnetic nanoparticle capture and poly-enzyme nanobead amplification for ultrasensitive detection and discrimination of DNA single nucleotide polymorphisms

The development of ultrasensitive methods for detecting specific genes and discriminating single nucleotide polymorphisms (SNPs) is important for biomedical research and clinical disease diagnosis. Herein, we report an ultrasensitive approach for label-free detection and discrimination of a full-match target-DNA from its cancer related SNPs by combining magnetic nanoparticle (MNP) capture and poly-enzyme nanobead signal amplification. It uses a MNP linked capture-DNA and a biotinylated signal-DNA to sandwich the target followed by ligation to offer high SNP discrimination: only the perfect-match target-DNA yields a covalently linked biotinylated signal-DNA on the MNP surface for subsequent binding to a neutravidin-horseradish peroxidase conjugate (NAV-HRP) for signal amplification. The use of polymer nanobeads each tagged with thousands of copies of HRPs greatly improves the signal amplification power, allowing for direct, amplifcation-free quantification of low aM target-DNA over a 6 order magnitude dynamic range (0.001-1000 fM). Moreover, this sensor also offers excellent discrimination between the perfect-match gene and its cancer-related SNPs and can positively detect 1 fM perfect-match target-DNA in the presence of 100 fold excess of co-existing SNPs. Furthermore, it works robustly in clinically relevant media (e.g. 10% human serum) and gives even higher SNP discrimination than that in clean buffers. This ultrasensitive SNP sensor appears to have excellent potential for rapid detection and diagnosis of genetic diseases.