A portable generic DNA bioassay system based on in situ oligonucleotide synthesis and hybridization detection

Abstract

In this study, we present a portable and generic DNA bioassay system based on in situ oligonucleotide synthesis followed by hybridization based detection. The system include two main parts, an oligonucleotide synthesizer and a fluorescence detection system. The oligonucleotide synthesizer is based on microfluidic technology and capable of synthesizing any desired oligonucleotide which can be either used as a primer for PCR based detection (external) or a probe for hybridization based detection (integrated) of a target DNA analyte. The oligonucleotide sequence can be remotely sent to the system. The integrated fluorescence detection system is based on a photodiode to detect Texas Red fluorophore as low as 0.5 fmol. The complete system, integrating the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The presented generic portable instrument has the potential to detect any desired DNA target sequence in the field. Potential applications are for homeland security and fast responses to emerging bio-threats.

Introduction

State of the art DNA based bioassays for the detection of biological warfare and infectious disease agents such as bacteria and viruses make use of pre-made oligonucleotides that function as either probe or primer (Ivnitski et al., 2003). For in-the-field application of these technologies, portability of such device is an important factor. DNA microarray technology has been proven to be a powerful tool for biological agents detection based on DNA hybridization (Heller, 2002, Stoughton, 2005). However, the expensive and complicated instrument setup made it difficult to be used for in-the-field applications. For other methods such as PCR based technologies, the devices can be fabricated in a very portable format. However, pre-made oligonucleotide primers with known sequence are used to carry out the assays (Kricka and Wilding, 2003, Neuzil et al., 2006). As a consequence, no immediate detection of emerging microbes or viruses or artificially manipulated or naturally mutated agents is possible. Therefore, there is an emerging need for technologies that can timely react to such threats.

Microfluidics is a promising technology due to its versatile use and ease of integration. The invention of microfluidic valves enabled microfluidic devices to manipulate and control fluids flow and therefore dramatically extend their use (Unger et al., 2000, Vieider et al., 1995). Oligonucleotide synthesis is one of such applications where sequential switching of a number of reagents was needed. As far as we know, just a few studies have been conducted on developing microfluidic device for oligonucleotide synthesis (Huang et al., 2007, Lee et al., 2010). However, no work has been done aiming to develop an oligonucleotide synthesizer in a portable format that has potential use for in-the-field applications.

Bioassay detection based on fluorescence such as DNA hybridization with fluorescence labeled targets is a well known powerful method. However, often expensive laser excitation sources and sophisticated detectors were used, which make them not suitable for in-the-field applications (Stoughton, 2005). With the development of LED technology, several studies demonstrated LEDs as cheap and small form factor light source often combined with photodiodes as detector (Li et al., 2005, Novak et al., 2006, Yang et al., 2009). In some studies, an oligonucleotide probe was directly immobilized on the photodiode surface, which limited the reusability of the detector (Li et al., 2005).

In this work, we present a portable and generic DNA bioassay system. The system is able to synthesize any desired oligonucleotide according to a remotely received sequence and consecutively used the oligonucleotide as a hybridization probe in its integrated fluorescence based detector to detect any target DNA in the field. The oligonucleotide synthesizer is based on microfluidic technology and the fluorescence detector is based on a LED light source and a photodiode detector. Alternatively the system can be used to synthesize oligonucleotide primers for PCR applications. Both primers and probes were synthesized by the portable system and their functionality was demonstrated by PCR and DNA hybridization experiments. The fluorescence detection system can detect the Texas Red fluorophore as low as 0.5 fmol and was successfully used for differentiating complementary and single-base mismatch DNA in hybridization experiments. The complete system, which integrated the oligonucleotide synthesizer and fluorescence detection system, was successfully used to distinguish DNA from two different bacteria strains. The system is portable and can synthesize DNA “on the run” with potential homeland security or remote analysis applications in-the-field.