A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.
A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.
Nanyang Technological University, 71 Nanyang Dr, 638075 Singapore. gslim1@e.ntu.edu.sg limgeoksoon@gmail.com.
Geok Soon
GS
Lim
Joseph S
JS
Chang
Zhang
Z
Lei
Ruige
R
Wu
Zhiping
Z
Wang
Kemi
K
Cui
Stephen
S
Wong
D000410:Alanine Transaminase / Q000235:genetics; D053000:Analytic Sample Preparation Methods / Q000295:instrumentation*; D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation; D056656:Lab-On-A-Chip Devices; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019095:Point-of-Care Systems; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D018511:Systems Integration; D018675:Toxicity Tests / Q000295:instrumentation*
D017931:DNA Primers; D012333:RNA, Messenger; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase