Jonas Høgberg

A lab-on-a-chip device for rapid identification of avian influenza viral RNA by solid-phase PCR

The endemic of Avian Influenza Virus (AIV) in Asia and epizootics in some European regions have caused serious economic losses. Multiplex reverse-transcriptase (RT) PCR has been developed to detect and subtype AIV. However, the number of targets that can be amplified in a single run is limited because of uncontrollable primer-primer interferences. In this paper, we describe a lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid-phase PCR on a microfluidic chip. A simple UV cross-linking method was used to immobilize the DNA probes on unmodified glass surface, which makes it convenient to integrate microarray with microfluidics. This solid-phase RT-PCR method combined RT amplification of extracted RNA in the liquid phase and species-specific nested PCR on the solid phase. Using the developed approach, AIV viruses and their subtypes were unambiguously identified by the distinct patterns of amplification products. The whole process was reduced to less than 1 hour and the sample volume used in the microfluidic chip was at least 10 times less than in the literature. By spatially separating the primers, highly multiplexed amplification can be performed in solid-phase PCR. Moreover, multiplex PCR and sequence detection were done in one step, which greatly simplified the assay and reduced the processing time. Furthermore, by incorporating the microarray into a microchamber-based PCR chip, the sample and the reagent consumption were greatly reduced, and the problems of bubble formation and solution evaporation were effectively prevented. This microarray-based PCR microchip can be widely employed for virus detection and effective surveillance in wild avian and in poultry productions.

Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay

DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.

Pre-storage of gelified reagents in a lab-on-a-foil system for rapid nucleic acid analysis

Reagent pre-storage in a microfluidic chip can enhance operator convenience, simplify the system design, reduce the cost of storage and shipment, and avoid the risk of cross-contamination. Although dry reagents have long been used in lateral flow immunoassays, they have rarely been used for nucleic acid-based point-of-care (POC) assays due to the lack of reliable techniques to dehydrate and store fragile molecules involved in the reaction. In this study, we describe a simple and efficient method for prolonged on-chip storage of PCR reagents. The method is based on gelification of all reagents required for PCR as a ready-to-use product. The approach was successfully implemented in a lab-on-a-foil system, and the gelification process was automated for mass production. Integration of reagents on-chip by gelification greatly facilitated the development of easy-to-use lab-on-a-chip (LOC) devices for fast and cost-effective POC analysis.

Gold Nanoparticles-Coated SU-8 for Sensitive Fluorescence-Based Detections of DNA

SU-8 epoxy-based negative photoresist has been extensively employed as a structural material for fabrication of numerous biological microelectro-mechanical systems (Bio-MEMS) or lab-on-a-chip (LOC) devices. However, SU-8 has a high autofluorescence level that limits sensitivity of microdevices that use fluorescence as the predominant detection workhorse. Here, we show that deposition of a thin gold nanoparticles layer onto the SU-8 surface significantly reduces the autofluorescence of the coated SU-8 surface by as much as 81% compared to bare SU-8. Furthermore, DNA probes can easily be immobilized on the Au surface with high thermal stability. These improvements enabled sensitive DNA detection by simple DNA hybridization down to 1 nM (a two orders of magnitude improvement) or by solid-phase PCR with sub-picomolar sensitivity. The approach is simple and easy to perform, making it suitable for various Bio-MEMs and LOC devices that use SU-8 as a structural material.

DNA microarrays immobilized on unmodified plastics in a microfluidic biochip for rapid typing of Avian Influenza Virus

Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on non-modified plastic surfaces. This one-step, cost-effective process provides very high immobilization and hybridization efficiencies and is applicable to many different types of polymers. Using this microarray fabrication technique, a portable cyclic olefin copolymer (COC) microarray device containing eight individually addressable microfluidic channels was developed for fast identification of Avian Influenza Virus (AIV) by DNA hybridization. This plastic biochip offers benefits of low fabrication cost and parallel processing of multiple samples, and could be used as a point-of-care device for clinical diagnostics and gene expression analysis.