= Biotechnology and Bioprocess Engineering 2007, 12: 634-639 Microfluidic Lab-on-a-chip for Microbial Identification on a DNA Microarray = eóìå=eç=iÉÉ N G=~åÇ=m~ìä=v~ÖÉê O = 1 Department of Chemical Engineering, Myongji University, Yongin 449-728, Korea 2 Department of Bioengineering, University of Washington, Seattle, WA 98195, USA ^Äëíê~Åí= A lab-on-a-chip for the rapid identification of microbial species has been developed for a water monitoring system. We em- ployed highly parallel DNA microarrays for the direct profiling of microbial populations in a sample. For the integration and minimization of the DNA microarray protocols for bacterial identification, rRNA was selected as a target nucleotide for probe:target hybridization. In order to hybridize target rRNA onto the probe oligonucleotide, intact rRNA extracted from bK= Åçäá rRNA was fragmented via chemical techniques in the lab-on-a-chip platform. The size of fragmented rRNA was less than 400 base pairs, which was confirmed by polyacrylamide gel electrophoresis. The fragmented rRNA was also labeled using fluorescent chemicals. The lab-on-a-chip for fragmentation and labeling includes a PDMS chaotic mixer for efficient mixing, operated by flow pressure. In addition, the fragmented rRNA was hybridized successfully on a DNA microarray with sample recirculation on a microfluidic platform. Our fragmentation and labeling technique will have far-reaching applications, which require rapid but complicated chemical genetic material processing on a lab-on-a-chip platform. © KSBB hÉóïçêÇëW =ãáÅêçÑäìáÇáÅ, ak^=ãÅáêç~êê~ó, ä~ÄJçåJ~JÅÜáé, ÅÜ~çíáÅ=ãáñÉê= = = = = fkqolar`qflk= A rapid microbial-detection system has an advantage over the existing time-consuming culture-based procedures for a variety of applications. The current approaches to microbial monitoring, for example in the clinical field, involve tradi- tional plate culturing techniques, which provide results in several days. In addition, these techniques are not sufficient for the comprehensive monitoring of all potentially problem- atic microbes [1]. Recently, DMS (DNA Microarray Sensor) technology has been used for rapid response time and a highly parallel analysis of target microbes [2]. For the effec- tive detection of target microbes with the DMS, PCR (Poly- merase Chain Reaction), or other target nucleotide amplify- cation schemes have been employed. However, these ampli- fication methods require more time and effort for their proc- esses [3]. In addition, the amplifications often introduce bi- ases for the quantification of natural bacterial populations. Bacterial 16S ribosomal RNA (rRNA) has been utilized as a target nucleotide for its identification. The level of sp- ecificity of the bacterial identification with 16S rRNA is sufficient for differentiation between very closely related microorganisms. Besides its specificity, rRNA itself is the G`çêêÉëéçåÇáåÖ=~ìíÜçê Tel: +82-31-330-6392 Fax: +82-31-337-1920 e-mail: hyunho@mju.ac.kr most abundant genetic material within microorganisms. Therefore, the use of naturally amplified rRNA in bacterial detection schemes would be clearly advantageous [4-6]. Typically, the purification of nucleic acids from speci- mens, most notably bacterial cultures, has traditionally been both laborious and time-consuming. Moreover, the typical method requires several pre-treatment and centrifugation steps [7]. However, there are many situations in which pre- treatment and centrifugation are not feasible, for example, when portable instruments must be utilized for analysis. Therefore, a substitute technique for pre-treatment and cen- trifugation should be devised, and a microfluidic system is currently the most promising candidate [3,7]. Microfluidics is a technology which enables many new experimental sciences, including microbiology [7-10]. Mi- crofluidics allows for the use of small volumes [7,8], avoids the need for reagents and reduces waste costs, and creates new types of assays which are impossible at the macroscopic scale. Recently, experimental designs involving the rapid fabrication of a versatile laminate-based technology have allowed for the integration of multiple detection processes on dedicated microfluidic systems [8]. The coupling of microfluidics with DNA microarrays us- ing rRNA is a more simple process than other more classi- cally used techniques, as it requires no additional PCR-based amplification of genetic materials. Instead, the size of rRNA targeted for the DNA microarray hybridization should be