Anant S. Gurung

Ultrasensitive nucleic acid biosensor based on enzyme–gold nanoparticle dual label and lateral flow strip biosensor

In this article, we describe an ultrasensitive nucleic acid biosensor (NAB) based on horseradish peroxidase (HRP)-gold nanoparticle (Au-NP) dual labels and lateral flow strip biosensor (LFSB). The results presented here expand on prior work (Mao et al., 2009a) by optimizing the preparation of HRP-Au-NP-DNA conjugates. It was found that sodium dodecyl sulfate (SDS) and the immobilization sequence of thiolated DNA and HRP on the Au-NP surface played very important roles to improve the sensitivity of the assay. After systematic optimization, the detection limit of current approach is 1000 times lower than that in prior work. Deposition of insoluble enzymatic catalytic product (red colored chromogen) on the captured Au-NPs at the test zone of LFSB offers a dramatic visual enhancement. Combining enzyme catalytic amplification with unique optical properties of Au-NPs, the NAB was capable of detecting of 0.01-pM target DNA without instrumentation. The NAB thus provides a rapid, sensitive, low-cost tool for the detection of nucleic acid samples. It shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

Visual detection of microRNA with lateral flow nucleic acid biosensor

We report a DNA-gold nanoparticle (DNA-GNP) based lateral flow nucleic acid biosensor for visual detection of microRNA (miRNA)-215 in aqueous solutions and biological samples with low-cost and short analysis time. Sandwich-type hybridization reactions among GNP-labeled DNA probe, miRNA-215 and biotin-modified DNA probes were performed on the lateral flow device. The accumulation of GNPs on the test zone of the biosensor enables the visual detection of miRNA-215. After systematic optimization, the biosensor was able to detect a minimum concentration of 60 pM miRNA-215. The biosensor was applied to detect miRNA-215 from A549 cell lysate directly without complex sample treatment, and the detection limit of 0.148 million cells was obtained. This study provides a simple, rapid, specific and low-cost approach for miRNA detection in aqueous solutions and biological samples, showing great promise for clinical application and biomedical diagnosis in some malignant diseases.

Carbon nanotube-based lateral flow biosensor for sensitive and rapid detection of DNA sequence.

In this article, we describe a carbon nanotube (CNT)-based lateral flow biosensor (LFB) for rapid and sensitive detection of DNA sequence. Amine-modified DNA detection probe was covalently immobilized on the shortened multi-walled carbon nanotubes (MWCNTs) via diimide-activated amidation between the carboxyl groups on the CNT surface and amine groups on the detection DNA probes. Sandwich-type DNA hybridization reactions were performed on the LFB and the captured MWCNTs on test zone and control zone of LFB produced the characteristic black bands, enabling visual detection of DNA sequences. Combining the advantages of lateral flow chromatographic separation with unique physical properties of MWCNT (large surface area), the optimized LFB was capable of detecting of 0.1 nM target DNA without instrumentation. Quantitative detection could be realized by recording the intensity of the test line with the Image J software, and the detection limit of 40 pM was obtained. This detection limit is 12.5 times lower than that of gold nanoparticle (GNP)-based LFB (0.5 nM, Mao et al. Anal. Chem. 2009, 81, 1660-1668). Another important feature is that the preparation of MWCNT-DNA conjugates was robust and the use of MWCNT labels avoided the aggregation of conjugates and tedious preparation time, which were often met in the traditional GNP-based nucleic acid LFB. The applications of MWCNT-based LFB can be extended to visually detect protein biomarkers using MWCNT-antibody conjugates. The MWCNT-based LFB thus open a new door to prepare a new generation of LFB, and shows great promise for in-field and point-of-care diagnosis of genetic diseases and for the detection of infectious agents.

Visual Detection of Hg2+ in Aqueous Solution Using Gold Nanoparticles and Thymine-Rich Hairpin DNA Probes

We report a sensitive method for visual detection of mercury ions (II) (Hg²⁺) in aqueous solution by using gold nanoparticles (Au-NPs) and thymine (T)-rich hairpin DNA probes. The thiolated hairpin DNA probe was immobilized on the Au-NP surface through a self-assembling method. Another thymine-rich, digoxin-labeled DNA probe was introduced to form DNA duplexes on the Au-NP surface with thymine-Hg²⁺-thymine (T-Hg²⁺-T) coordination in the presence of Hg²⁺. The Au-NPs associated with the formed duplexes were captured on the test zone of a lateral flow strip biocomponent (LFSB) by immunoreaction events between the digoxin on the duplexes and anti-digoxin antibodies on the LFSB. The accumulation of Au-NPs produced a characteristic red band on the test zone, enabling visual detection of Hg²⁺ without instrumentation. A detection limit of 0.1 nM was obtained under optimal experimental conditions. This method provides a simple, rapid, sensitive approach for the detection of Hg²⁺ and shows great promise for point-of-care and in-field detection of environmentally toxic mercury.

Visual Detection of Gene Mutations Based on Isothermal Strand-Displacement Polymerase Reaction and Lateral Flow Strip

Here, we describe a simple and sensitive approach for visual detection of gene mutations based on isothermal strand-displacement polymerase reactions (ISDPR) and lateral flow strip (LFS). The concept was first demonstrated by detecting the R156H-mutant gene of keratin 10 in Epidermolytic hyperkeratosis (EHK). In the presence of biotin-modified hairpin DNA and digoxin-modified primer, the R156H-mutant DNA triggered the ISDPR to produce numerous digoxin- and biotin-attached duplex DNA products. The product was detected on the LFS through dual immunoreactions (anti-digoxin antibody on the gold nanoparticle (Au-NP) and digoxin on the duplex, anti-biotin antibody on the LFS test zone and biotin on the duplex). The accumulation of Au-NPs produced the characteristic red band, enabling visual detection of the mutant gene without instrumentation. After systematic optimization of the ISDPR experimental conditions and the parameters of the assay, the current approach was capable of detecting as low as 1-fM R156H-mutant DNA within 75 min without instrumentation. Differentiation of R156H- and R156C-mutant DNA on the R156 mutation site was realized by using fluorescein- and biotin-modified hairpin probes in the ISDPR process. The approach thus provides a simple, sensitive, and low-cost tool for the detection of gene mutations.