Multiplexed Bead-Based Mesofluidic System for Gene Diagnosis and

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Anal. Chem. 2010, 82, 9925–9931

Multiplexed Bead-Based Mesofluidic System for Gene Diagnosis and Genotyping Sheng-Quan Jin,† Bang-Ce Ye,*,† Hao Huo,† Ai-Jun Zeng,‡ Cheng-Ke Xie,‡ Bing-Qiang Ren,‡ and Hui-Jie Huang‡ Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China, and Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Shanghai 201800, China We have developed a novel multiplexed bead-based mesofluidic system (MBMS) based on the specific recognition events on the surface of a series of microbeads (diameter 250 µm) arranged in polydimethylsiloxane (PDMS) microchannels (diameter 300 µm) with the predetermined order and assembled an apparatus implementing automatically the high-throughput bead-based assay and further demonstrated its feasibility and flexibility of gene diagnosis and genotyping, such as β-thalassemia mutation detection and HLA-DQA genotyping. The apparatus, consisting of bead-based mesofluidic PDMS chip, liquid-processing module, and fluorescence detection module, can integrate the procedure of automatedsampling, hybridization reactions, washing, and in situ fluorescence detection. The results revealed that MBMS is fast, has high sensitivity, and can be automated to carry out parallel and multiplexed genotyping and has the potential to open up new routes to flexible, high-throughput approaches for bioanalysis. The completion of the Human Genome Project has inspired new efforts in the area of mutation detection and genotyping for gene diagnostics. Gene analysis is very critical to clinical diagnosis, for the essential of individual inherited difference is on the gene level, and is more accurate than serotyping and cell typing. Conventional methods used for gene diagnosis include restriction fragment length polymorphism (RFLP),1,2 reverse dot blot hybridization (RDB),3-6 and Southern blot.7 Most of these methods are relatively complex, time-consuming, and cumbersome. In the past decade, DNA microarray become a powerful tool for rapid * To whom correspondence should be addressed. Phone: 0086-21-64252094. Fax: 0086-21-64252094. E-mail: [email protected]. † East China University of Science & Technology. ‡ Chinese Academy of Sciences. (1) Ota, M.; Seki, T.; Fukushima, H.; Tsuji, K.; Inoko, H. Tissue Antigens 1992, 39, 187–202. (2) Old, J. M.; Petrou, M.; Modell, B.; Weatherall, D. J. Br. J. Haematol. 1984, 57, 255–263. (3) Sutcharitchan, P.; Saiki, R.; Huisman, T. H.; Kutlar, A.; McKie, V.; Erlich, H.; Embury, S. H. Blood 1995, 86, 1580–5. (4) Buyse, I.; Decorte, R.; Baens, M.; Cuppens, H.; Semana, G.; Emonds, M. P.; Marynen, P.; Cassiman, J. J. Tissue Antigens 1992, 41, 1–14. (5) Cai, S. P.; Wall, J.; Kan, Y. W.; Chehab, F. F. Hum. Mutat. 1994, 3, 59–63. (6) Li, D. Z.; Liao, C.; Li, J.; Huang, Y. N.; Xie, X. M.; Wei, J. X.; Wu, S. Q. Hemoglobin 2006, 30, 365–370. (7) Old, J.; Petrou, M.; Varnavides, L.; Layton, M.; Modell, B. Prenatal Diagn. 2000, 20, 986–991. 10.1021/ac1024792  2010 American Chemical Society Published on Web 10/29/2010

and high-throughput detection of genes and has been applied successfully for gene diagnosis.8-14 However traditional microarrays have either limitations with respect to slow reaction kinetics or in certain cases difficult integration and automation of operations. Recently much effort has been devoted to develop beadbased assays where encoded micrometer-sized beads are used for attaching probe biomolecules. The suspension assay using microbeads provides a powerful, flexible, and high-throughput platform, which offers many advantages over alternative methods. Methods for multiplexing such assays involve encoding the beads, either spatially (as is the case in a planar microarray) or by spectra (such as fluorescence spectrum, Raman spectrum, reflection spectrum, or absorbance spectrum),15-17 graphical pattern18 or barcode tags.19,20 A recent review gave the detailed discussion about bead-encoding technologies.21 So far, numerous wellestablished bead-based assays have been developed for sequencing, gene expression, and genotyping in the past decade. Sydney Brenner et al. described a gene expression analysis approach by massively parallel signature sequencing (MPSS) on microbead arrays.22 Millions of template-containing microbeads (diameter 5 µm), assembling a planar array in a microfluidic chamber, can be simultaneously tracked through successive cycles of ligation, probing, and cleavage to generate a time series of spatially (8) Ye, B. C.; Zhang, Z. F.; Lei, Z. S. Genet. Test. 2007, 11, 75–83. (9) Lu, Y.; Kham, S. K. Y.; Tan, P. L.; Quah, T. C.; Heng, C. K.; Yeoh, A. E. J. Genet. Test. 2005, 9, 212–219. (10) van Moorsel, C. H. M.; van Wijngaarden, E. E.; Fokkema, I.; den Dunnen, J. T.; Roos, D.; van Zwieten, R.; Giordano, P. C.; Harteveld, C. L. Eur. J. Hum. Genet. 2004, 12, 567–573. (11) Chan, K. M.; Wong, M. S.; Chan, T. K.; Chan, V. Br. J. Haematol. 2004, 124, 232–239. (12) Lee, K. R.; Park, E.; Moon, S. H.; Kim, J. M.; Kwon, O. J.; Kim, M. H.; Sohn, Y. H.; Ko, S. Y.; Oh, H. B. Tissue Antigens 2008, 72, 568–577. (13) Zhang, F.; Hu, S. W.; Huang, J.; Wang, H.; Wen, Z.; Geng, Y. Y.; Wang, S. Q. Pharmacogenomics 2006, 7, 973–985. (14) Zhang, F.; Hu, S. W.; Huang, J.; Wang, H.; Zhang, W.; Geng, Y. Y.; Wang, S. Q. Tissue Antigens 2005, 65, 467–473. (15) Pregibon, D. C.; Toner, M.; Doyle, P. S. Science 2007, 315, 1393–1396. (16) Eastman, P. S.; Ruan, W.; Doctolero, M.; Nuttall, R.; de Feo, G.; Park, J. S.; Chu, F.; Cooke, P.; Gray, J. W.; Li, S.; Chen, F. F. Nano Lett. 2006, 6, 1059–1064. (17) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631–635. (18) Zhi, Z.; Morita, Y.; Hasan, Q.; Tamiya, E. Anal. Chem. 2003, 75, 4125– 4131. (19) Li, Y.; Thi, Y.; Luo, D. Nat. Biotechnol. 2005, 23, 885–889. (20) Geiss, G. K.; Bumgarner, R. E.; Birditt, B.; Dahl, T.; Dowidar, N.; Dunaway, D. L.; Fell, H. P.; Ferree, S.; George, R. D.; Grogan, T.; James, J. J.; Maysuria, M.; Mitton, J. D.; Oliveri, P.; Osborn, J. L.; Peng, T.; Ratcliffe, A. L.; Webster, P. J.; Davidson, E. H.; Hood, L. Nat. Biotechnol. 2008, 26, 317–325. (21) Birtwell, S.; Morgan, H. Integr. Biol. 2009, 1, 345–362.

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localized signals for gene expression. Illumina has also developed a multiplex automated bead-based platform for gene expression profiling and genotyping. The beads (diameter 3 µm) with ∼80 bp oligonucleotides, consisting of an address code (∼30 bp) and a 50 bp gene-specific sequence, are randomly deposited into cavities on a glass slide. The resultant array can be decoded to determine which bead is in which cavity.23,24 Combined with the microfluidic device, these automated bead-based arrays platforms offer high capability for multiplexing assays, particularly well suited for basic research of functional genomics in some sophisticated laboratories. However, customized bead-microarrays with limited multiplexing capability, focusing on only small groups of specific desirable SNP or gene panels, are in demand in more and more research laboratories and clinics to meet their specific purposes, especially when the SNP or gene panels have clinical applications. In fact, a large number of specific SNP or gene sets can be employed for clinical diagnosis and routine clinical practices. Generally, these bead-based arrays with lower encoding capacities (approximately 102 codes) may often be sufficient for genetic diagnosis and immunoassays and have advantages not only in their flexibility in SNP and gene inclusion but also in their low cost for fabrication and operation. Furthermore, application-targeted bead-arrays in microfluidic or mesofluidic channels can easily be integrated into devices for biochemical research and point-of-care medical diagnostic applications (such as Luminex Corporation’s xMAP using 100 color-encoded beads).25,26 However, fluorescence spectrum encoding method has some limitations: dyes tend to be quenched or bleached, and the fluorescence of beads can interfere with the signal from detection reaction. Very recently, we developed a novel design strategy of multiplexed bead-based mesofluidic system (MBMS) for bioanalysis. We exploited the mesofluidic system based on the specific recognition event on the surface of a series of microbeads arranged in PDMS microchannels to achieve multiplexing detection without the need for color-coding. The systems have been successfully applied for determination of foodborne pathogens27 and drug residues.28 Although more sample volume (∼10 µL) is needed, mesofluidic chip (channel diameter, >100 µm) analysis provides better fluidics control and maneuverability than the microfluidic system (channel diameter, G) point mutation of the HBB gene as a model. A pair of probes was designed for -28(A > G) site, one complementary to the wild sequence (-28W) and one to the variant (-28M). In the analysis of point mutation and genotyping, it is assumed that the strength of fluorescent signal of each microbead is representative of the amount of labeled DNA associated with that microbead. The amount of labeled DNA on each microbead relies upon the stability of the duplex between labeled DNA and probe immobilized on that microbead. The wildto mutant signal ratios (W/M), wild type probe (denoted as W) to mutant probe (denoted as M) can be employed to detect the genotypes. For heterozygous samples, two oligonucleotide probes in a group could be perfectly matched with the PCR product, each matched with a different allele. For homozygous samples, only one probe in each group would be perfectly matched with the PCR product. The probes should be carefully selected to achieve significant discrimination of single-base mismatch. The dilutions of the HBB gene PCR product were used to investigate the sensitivity of the discrimination of point mutations. Figure 2D showed a correlation of the signal intensity with the concentration of PCR targets. The fluorescence intensity discriminated the point mutation evident at 10-11 M, and the mutant-towild signal ratios exceeded 5:1. The data demonstrated that MBMS has the ability to resolve single-base mismatches at the concentration of 10 pM, which had the similar sensitivity as the fiber-optic DNA random microbead (diameter 3.1 µm) array with dye-encoding (detection limit of 10 pM in 30 min).32 Recently, Zhang et al. described a novel microfluidic device with a microbead array for genotyping HBV using quantum dot as labels, and the sensitivity was estimated to 4 pM.35 However, the microfluidic system without microbeads, in which probes were directly immobilized on the slide surface in microchannels, only exhibited the sensitivity of ∼10 nM (1.4 ng/µL PCR product of 260 bp) for gene detection.36 Furthermore, a series of five repetitive measurements with 3 µM DNA target was used for investigating the accuracy of the bead-based mesofluidic system and obtained a coefficient of variation (CV) of ∼10%, demonstrating an excellent reproducibility of the assay. Detection of β-Thalassamia Mutation. The β-thalassemia results from more than 200 different HBB gene mutations. Despite this marked molecular heterogeneity, the prevalent molecular defects are limited in each at-risk population. In south China, eight mutations usually account for the large majority of HBB diseasecausing alleles, including -28(A > G), -29(A > G), β17(A > T), HbE26(G > A), IVS(I-1)(G > T), β(41-42)(delTCTT), β43(G > T), IVS(II-654)(C > T). The probe set corresponding to these mutations was designed and optimized. Some clinical samples and (33) Situma, C.; Hashimoto, M.; Soper, S. A. Biomol. Eng. 2006, 23, 213–231. (34) Wei, C. W.; Cheng, J. Y.; Huang, C. T.; Yen, M. H.; Young, T. H. Nucleic Acids Res. 2005, 33, e78. (35) Zhang, H.; Xu, T.; Li, C. W.; Yang, M. S. Biosens. Bioelectron. 2010, 25, 2402–2407. (36) Wang, L.; Li, P. C. J. Agric. Food Chem. 2007, 55, 10509–10516.

Table 1. The Allele Frequence of β-Thalassemia Mutation in All 40 Samples mutations

heterozygote

homozygote

-28 -29 β17 HbE26 IVS(I-1) β(41-42) β43 IVS(II-654) total wild type

6 1 2 2 1 8 1 3 24

1 0 0 0 0 0 0 0 1 13

compound heterozygote

allele frequence

0 1 1 0 0 0 1 1 4(÷2)

25.9% 7.4% 11.1% 7.4% 3.7% 29.6% 7.4% 14.8% 27 13

eight plasmids (as homozygous samples) were used as the known samples to validate the new system. Every sample was tested in triplicate. The results show high fluorescent signal on the microbeads containing the probes, which perfectly match to target gene sequence. Very weak fluorescent signal is found on the microbeads containing related probes, which single-base mismatch to corresponding positions. All results were correct, as compared with the known sequence, and W/M (or M/W) signal ratios mostly exceeded 5:1, which can be considered as a threshold to detect and discriminate mutations. For the detection of point mutation of blood samples, the W/M signal ratios obtained produced unequivocal assignment of samples genotype. All probes in each pair/group served as the control for each other, offering unequivocal and specific discrimination of each genotype. To demonstrate how the MBMS system could be used to genotype blind samples, we conducted an equivalency study of 40 clinical samples whose HBB genes were sequenced. In all sets of experiments for each mutation, the W/M ratios were employed to distinguish single-base mismatch, allowing assignment of wildtype, heterozygous, or homozygous status to each sample as expected. No false-positive or false-negative result was observed in any case. The MBMS detection results were coincident with standard sequencing. Among the 40 samples, 26 mutant heterozygotes (2 double-heterozygotes) and 1 mutant homozygote were found. β(41-42)(delTCTT) was found in 8 out of the 40 (29.6%), the total number of β-thalassemia mutated alleles, followed by 7 cases of -28(A > G) (25.9%). The relative frequencies of the different thalassemia genotypes of all 40 samples are shown in Table 1. Genotyping of HLA-DQA1 Gene. As a further illustration of the utility of the MBMS, the genotyping of HLA-DQA1 was investigated. There are 28 HLA-DQA genotypes (including subgenotypes) according to gene sequence of DQA exon 2 from the IMGT/HLA sequence database (release 2.11.0). A total of 31 oligonucleotide probes were designed for all polymorphisms in the second exon of the DQA gene (Table S2 in the Supporting Information). The probes were carefully selected to achieve a significant discrimination of single-base mismatch. All genotypes and hybridization patterns of all probes were listed in Figure 3. All probes were divided into 10 groups from A group to J group; a gray box indicated a positive signal of hybridization, and a white box indicated a negative signal of hybridization. Because of the identical gene sequence in the target region, some subgenotypes could not be differentiated, which were DQA*010101, DQA*010102, DQA*010401, DQA*010402, and DQA*0105; DQA*010201 andDQA*010202;DQA*030101,DQA*0302,andDQA*0303;DQA*040101, Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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Figure 3. Hybridization signal patterns of all 30 probes for all HLA-DQA genotypes. Black boxes indicate positive signals of hybridization, and white boxes indicate negative signals.

DQA*040102, DQA*0402, DQA*0403N, and DQA*0404; DQA*050101, DQA*050102, DQA*0503, and DQA*0505; DQA*060101 and DQA*0602. Although the method does not perform DQA high-resolution genotyping, it could successfully differentiate DQA specificity. Moreover, the resolution could be easily developed by adding new probe sequences descriptive of novel polymorphisms without significantly increased costs. The microbeads precoated with 31 probes were sequenced in the microchannel. The PCR product (229 bp) of the second exon of the DQA1 gene was injected into the microchannel and hybridized with probes on the surface of the beads. Genotypes of DQA were identified by the positive and negative signals in each probe group. For a homozygous sample, only one probe in each group would be perfectly matched to the PCR products; for heterozygous one, there were two positive signals in a group and each matched with a different allele. The highest signal(s) in each group was chosen as the positive signal(s), and an intensity ratio of 3:1 was employed as the threshold to categorize the positive and negative signals. Generally, the intensity ratio of a positive signal(s) and negative signal(s) in each group exceeded 3:1, which depend on the physical characteristics of the probes, conditions of hybridization, and washing. As a heterozygote, the threshold was decided as smaller than 1.5. After identification of positive signal(s) in each group, compared with hybridization signal patterns of HLA-DQA genotypes (Figure 3), the matched genotype(s) was the genotype(s) of the sample. A total of 32 samples, whose DQA1 alleles had been typed previously by direct sequencing, were tested using the MBMS method. All genomic DNA were derived from peripheral blood lymphocyte of irrelative individuals. The positive probes of the sample were categorized according to the threshold, and the genotype of the sample was determined from the hybridization signal patterns of HLA-DQA genotypes. The results of 31 samples 9930

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were completely consistent with the direct sequencing results. Only one heterozygous sample was determined as a homozygote by MBMS. Also, most frequency genotypes are DQA*03 (including DQA*030101, DQA*0302, and DQA*0303) and DQA*05 (including DQA*050101, DQA*050102, DQA*0503, and DQA*0505), which is in accordance with DQA1 genotypes frequencies of the population in China. This equivalency study demonstrated that the MBMS method could get satisfactory results to these heterozygous combinations and homozygous samples within the

Figure 4. The analysis of hybridization data for sample DQA1*0311. Each peak corresponds to one microbead above, which was immobilized with the corresponding probe.

medium resolution. The results of analysis of sample DQA1*0311 was shown in Figure 4. CONCLUSIONS In this study, a MBMS system was developed, which contains a bead-based mesofluidic PDMS chip, liquid-processing module, and fluorescence detection module, and integrated automatedsampling, hybridization reactions, washing, and in situ fluorescence scanning. The combination of a probe-modified microbeads array in mesofluidic devices should give new, flexible, highthroughput approaches. With this MBMS, all of the analysis procedure can be carried out automately, and the whole of the analysis could be done within an hour. All of the process was conducted in microchannels, which reduce the sample volume and avoid the liquid evaporation and cross contamination. This method is fast, sensitive, and high-throughput and can be carried out automatically. With this MBMS, the analysis of β-thalassemia and genotyping of HLA-DQA were performed and have a good coincidence with standard sequencing. It is demonstrated that MBMS has the capability to be applied for the effective gene

diagnosis and has the potential in biological fundamental research, food safety, and environmental monitoring. ACKNOWLEDGMENT This work was financially supported by NSF (20627005, 21075040), the Shanghai Project (Grant 09JC1404100), the SKLBE (Grant 2060204), Program for New Century Excellent Talents (Grant NCET-07-0287), and the Fundamental Research Funds for the Central Universities. SUPPORTING INFORMATION AVAILABLE Addition information on the sequences and names of probes and primers for β-thalassemia analysis and the sequences and names of probes for HLA-DQA gene typing. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 8, 2010. Accepted October 18, 2010. AC1024792

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