JBB : Journal of Bioscience and Bioengineering

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Journal of Bioscience and Bioengineering vol.118 cover

 

Journal of Bioscience and Bioengineering – Recent Articles

  • Binding affinity of ssDNA is improved by attachment of dsDNA regions
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Hiroyuki Ida , Akira Tachibana , Toshizumi Tanabe

    LidNA, a microRNA inhibitor consisting of a microRNA binding ssDNA region sandwiched between dsDNA regions had higher affinity to target oligonucleotides than that without dsDNA region. This enhancement in affinity was found to be owing to the suppressed mobility of ssDNA region by the presence of dsDNA regions.





  • Co-expression of a heat shock transcription factor to improve conformational quality of recombinant protein in Escherichia coli
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Shao-Yen Hsu , Yu-Sheng Lin , Shu-Jyuan Li , Wen-Chien Lee

    A co-expression system was established in Escherichia coli for enhancing the cellular expression of heat shock transcription factor, sigma 32 (σ32). A Shine–Dalgarno sequence and the rpoH gene of E. coli, which encodes σ32, were cloned into a bacterial plasmid containing a gene fusion encoding a doubly tagged N-acetyl-d-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R). After the IPTG induction, a substantially higher level of sigma 32 was observed up to 3 h in the co-expression cells, but an enhancement in the solubility of target protein was manifest only in the first hour. Nevertheless, the co-expression of sigma 32 led to higher level of Neu5Ac aldolase enzymatic activity in both the soluble and insoluble (inclusion body) fractions. The Neu5Ac aldolase activity of the supernatant from the lysate of cells co-expressing GST-Neu5Ac aldolase-5R and recombinant σ32 was 3.4-fold higher at 3 h postinduction than that in cells overexpressing GST-Neu5Ac aldolase-5R in the absence of recombinantly expressed σ32. The results of acrylamide quenching indicated that the conformational quality of the fusion protein was improved by the co-expression of recombinant σ32. Thus, the increased level of intracellular σ32 might have created favorable conditions for the proper folding of recombinant proteins through the cooperative effects of chaperones/heat shock proteins expressed by the E. coli host, which resulted in smaller inclusion bodies, improved conformational quality and a higher specific activity of the overexpressed GST-Neu5Ac aldolase-5R protein.





  • Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Yi Liu , Wenjing Cui , Zhongmei Liu , Youtian Cui , Yuanyuan Xia , Michihiko Kobayashi , Zhemin Zhou

    Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase.





  • Analysis of specific proteolytic digestion of the peptidoglutaminase–asparaginase of koji molds
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Kotaro Ito , Yasuji Koyama

    AsGahB, a peptidoglutaminase–asparaginase acting as the main glutaminase in Aspergillus sojae, was previously purified from the cytoplasm of overexpressing strains. Here, we found that specific proteolytic digestion of AsGahB by extracellular proteases of koji molds is similar to that of AsGahA which exists in proteolytic form under solid-state culture.





  • Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Akkaraphol Srichaisupakit , Takao Ohashi , Kazuhito Fujiyama

    Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation.





  • Importance of glucose-6-phosphate dehydrogenase (G6PDH) for vanillin tolerance in Saccharomyces cerevisiae
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Trinh Thi My Nguyen , Sakihito Kitajima , Shingo Izawa

    Vanillin is derived from lignocellulosic biomass and, as one of the major biomass conversion inhibitors, inhibits yeast growth and fermentation. Vanillin was recently shown to induce the mitochondrial fragmentation and formation of mRNP granules such as processing bodies and stress granules in Saccharomyces cerevisiae. Furfural, another major biomass conversion inhibitor, also induces oxidative stress and is reduced in an NAD(P)H-dependent manner to its less toxic alcohol derivative. Therefore, the pentose phosphate pathway (PPP), through which most NADPH is generated, plays a role in tolerance to furfural. Although vanillin also induces oxidative stress and is reduced to vanillyl alcohol in a NADPH-dependent manner, the relationship between vanillin and PPP has not yet been investigated. In the present study, we examined the importance of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the rate-limiting NADPH-producing step in PPP, for yeast tolerance to vanillin. The growth of the null mutant of G6PDH gene (zwf1Δ) was delayed in the presence of vanillin, and vanillin was efficiently reduced in the culture of wild-type cells but not in the culture of zwf1Δ cells. Furthermore, zwf1Δ cells easily induced the activation of Yap1, an oxidative stress responsive transcription factor, mitochondrial fragmentation, and P-body formation with the vanillin treatment, which indicated that zwf1Δ cells were more susceptible to vanillin than wild type cells. These findings suggest the importance of G6PDH and PPP in the response of yeast to vanillin.





  • Role of Karanja deoiled cake based medium in production of protease and fatty acids by Paecilomyces lilacinus 6029
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Abhishek Sharma , Satyawati Sharma , Savita Yadav , Satya N. Naik

    Protease and fatty acids are known to serve as pathogenic factors against root-knot nematodes. Here, we utilized Karanja deoiled cake as a nitrogen source for the first time in induction of protease by Paecilomyces lilacinus. Fatty acids, especially butyric acid, have also been detected in the same fungal culture filtrate.





  • Molecular analysis of Oenococcus oeni and the relationships among and between commercial and autochthonous strains
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Lucía González-Arenzana , Rosa López , Javier Portu , Pilar Santamaría , Teresa Garde-Cerdán , Isabel López-Alfaro

    The presence and distribution of genotypes from malolactic starter cultures between the autochthonous microbiota in fermenting Rioja wines have been studied in this paper. The commercial cultures characterization allowed to identify different species and common pulsed field gel electrophoresis and randomly amplified polymorphic DNA genotypes in several brands of Oenococcus oeni starter cultures. Four indistinguishable genotypes were found between the commercial and the autochthonous O. oeni strains. These four genotypes appeared during different vintages in seven of the 10 sampled wineries despite bacterial starter cultures had never been used. Therefore, the detection of commercial LAB genotypes indistinguishable from the autochthonous ones involved their removal from a selection process. These genotypes represented the 17.7% of autochthonous O. oeni isolated in this region during three consecutive years. This fact demonstrated that performing similar studies as previous selection criteria is advisable to avoid large work and the proposal of one commercialized bacteria.





  • Effect of ammonium nitrogen concentration on the ammonia-oxidizing bacteria community in a membrane bioreactor for the treatment of anaerobically digested swine wastewater
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Qianwen Sui , Chong Liu , Hongmin Dong , Zhiping Zhu

    A membrane bioreactor (MBR) was developed for the treatment of anaerobically digested swine wastewater and to investigate the effect of ammonium nitrogen concentration on biological nitrogen removal and ammonia-oxidizing bacteria (AOB) community structures. The MBR achieved a high NH4 +-N removal efficiency of 0.08 kgNMLSS−1d−1 and removed 95% of the influent NH4 +-N. The TN removal rate was highest of 82.62% at COD/TN and BOD5/TN ratios of 8.76 ± 0.30 and 3.02 ± 0.09, respectively. With the decrease in ammonium nitrogen concentrations, the diversity of the AOB community declined and showed a simple pattern of DGGE. However, the AOB population size remained high, with abundance of 107–109 copies mL−1. With the decrease of ammonium nitrogen concentrations, Nitrosomonas eutropha gradually disappeared, whereas Nitrosomonas sp. OZK11 showed constant adaptability to survive during each treatment stage. The selective effect of ammonium concentration on AOB species could be due to the affinity for NH4 +-N. In this study, the changes of ammonium nitrogen concentrations in digested swine wastewater were found to have selective effects on the composition of AOB community, and biological nitrogen removal was improved by optimising the influencing parameters.





  • Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted pharmaceutical wastewater
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Zhu Chen , Yuguang Wang , Kai Li , Hongbo Zhou

    The performance of an up-flow anaerobic sludge blanket (UASB) reactor was investigated in the treatment of diluted pharmaceutical fermentation wastewater for a continuous operation of 140 days. The dynamics and compositions of the microbial community were monitored using polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis. Increase of the organic loading rate (OLR) from 2.7 kg COD/m3 d to 7.2 COD/m3 d led to an increase in the COD removal efficiency from 83% to 91%. The dominant bacteria shifted from Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%) to Firmicutes (48.4%), Bacteroidetes (9.5%) and Proteobacteria (5.4%). For archeaon, the dominant groups changed from Thermoplasmata (24.4%), Thermoprotei (18.0%) and Methanobacteria (30.8%) to Thermoplasmata (70.4%) and Methanomicrobia (16.8%). Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could outcompete other species and dominated in the reactor under higher OLR. The results indicated that, to some extent, microbial community shift could reflect the performance of the reactor and a significant community shift corresponded to a considerable process event.





  • Abundance, transcription levels and phylogeny of bacteria capable of nitrous oxide reduction in a municipal wastewater treatment plant
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Kang Song , Toshikazu Suenaga , Aki Hamamoto , Kouichi Satou , Shohei Riya , Masaaki Hosomi , Akihiko Terada

    Nitrous oxide (N2O) production and expression of genes capable of its reduction were investigated in two full-scale parallel plug-flow activated sludge systems. These two systems continuously received wastewater with the same constituents, but operated under distinct nitrification efficiencies due to mixed liquor suspended solid (MLSS) concentration and the different hydraulic retention times (HRTs). A shorter HRT in system 2 resulted in a lower nitrification efficiency (40–60%) in conjunction with a high N2O emission (50.6 mg-N/L/day), whereas there was a higher nitrification efficiency (>99%) in system 1 with low N2O emission (22.6 mg-N/L/day). The DNA abundance of functional genes responsible for nitrification and denitrification were comparable in both systems, but transcription of nosZ mRNA in the lower N2O emission system (system 1) was one order of magnitude higher than that in the higher N2O emission system (system 2). The diversity and evenness of the nosZ gene were nearly identical; however, the predominant N2O reducing bacteria were phylogenetically distinct. Phylogenetic analysis indicated that N2O-reducing strains only retrieved in system 1 were close to the genera Rhodobacter, Oligotropha and Shinella, whereas they were close to the genera Mesorhizobium only in system 2. The distinct predominant N2O reducers may directly or indirectly influence N2O emissions.





  • Orthogonal array deciphering MRS medium requirements for isolated Lactobacillus rhamnosus ZY with cell properties characterization
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Yu Zhang , I-Son Ng , Chuanyi Yao , Yinghua Lu

    Lactobacillus rhamnosus is a well-known lactic acid bacterium (LAB), but a new ZY strain was isolated for the first time from commercial probiotic powder recently. Although many studies have focused on developing cost-effective media for the production of LAB, the de Man, Rogosa and Sharpe (MRS) medium is still the most common medium for bioprocesses. The aim of the current study is to decipher the composition of MRS based on a statistical approach, which will allow a higher biomass of Lactobacillus to be obtained. In Taguchi's approach, an L27 orthogonal array was adopted to evaluate the significance of 10 ingredients in MRS, in which the effects of the components were ranked according to their effect on biomass at OD600 as dextrose > MnSO4·H2O > beef extract > CH3COONa > MgSO4 > yeast extract > proteose peptone > K2HPO4 > ammonium citrate > Tween 80. Although the individual trace elements of ammonium citrate, K2HPO4, CH3COONa and MgSO4 in MRS had an insignificant influence on the biomass after statistical analysis, the total elimination of trace elements would predominantly affect the cell growth of Lactobacillus. Further characterization of the cell properties through attenuated total reflectance of Fourier transform infrared (ATR-FTIR) spectroscopy and protein identification via SDS-PAGE coupled with tandem mass spectrometry implied that dextrose as major carbon source in MRS played the most crucial role for L. rhamnosus production.





  • Efficient production of mutant phytase (phyA-7) derived from Selenomonas ruminantium using recombinant Escherichia coli in pilot scale
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): John Chi-Wei Lan , Chih-Kai Chang , Ho-Shing Wu

    A mutant gene of rumen phytase (phyA-7) was cloned into pET23b(+) vector and expressed in the Escherichia coli BL21 under the control of the T7 promoter. The study of fermentation conditions includes the temperature impacts of mutant phytase expression, the effect of carbon supplements over induction stage, the inferences of acetic acid accumulation upon enzyme expression and the comparison of one-stage and two-stage operations in batch mode. The maximum value of phytase activity was reached 107.0 U mL−1 at induction temperature of 30°C. Yeast extract supplement demonstrated a significant increase on both protein concentration and phytase activity. The acetic acid (2 g L−1) presented in the modified synthetic medium demonstrated a significant decrease on expressed phytase activity. A two-stage batch operation enhanced the level of phytase activity from 306 to 1204 U mL−1 in the 20 L of fermentation scale. An overall 3.7-fold improvement in phytase yield (35,375.72–1,31,617.50 U g−1 DCW) was achieved in the two-stage operation.





  • Characterization of human papillomavirus 6b L1 virus-like particles isolated from silkworms using capillary zone electrophoresis
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Thomas Hahne , Muthukutty Palaniyandi , Tatsuya Kato , Peter Fleischmann , Hermann Wätzig , Enoch Y. Park

    Human papillomavirus 6b L1 virus-like particles (VLPs) were successfully expressed using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system and rapidly purified using size exclusion chromatography after ultracentrifugation procedure and characterized by capillary zone electrophoresis (CZE). The average capillary electrophoresis migration time was 11 min with the relative standard deviation (RSD) of 0.3% of human papillomavirus 6b L1 VLPs. After this threefold fractionation, the CZE samples were still further investigated by dynamic light scattering and immuno blotting. The versatile technique, CZE not only proved to be a valuable tool for VLP characterization, but was also found to be reliable and precise. Thus CZE will also be an important option for the quality control of VLPs in pharmaceutical research level.





  • Control of adhesion of human induced pluripotent stem cells to plasma-patterned polydimethylsiloxane coated with vitronectin and γ-globulin
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Ryotaro Yamada , Koji Hattori , Saoko Tachikawa , Motohiro Tagaya , Toru Sasaki , Shinji Sugiura , Toshiyuki Kanamori , Kiyoshi Ohnuma

    Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices.





  • Keratinization induced by air exposure in the reconstructed human epidermal model: An in vitro model of a cultured epithelial autograft
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Takao Hanada , Yuichi Itahara , Masakazu Katoh , Masukazu Inoie , Ken-ichiro Hata

    A reconstructed human epidermis, an in vitro model of a cultured epithelial autograft, was used to examine the formation of a stratum corneum induced by exposure to air. A prolonged wet condition and excess application of petrolatum on the dressing reduced efficient production of the stratum corneum.





  • Microfluidic perfusion culture chip providing different strengths of shear stress for analysis of vascular endothelial function
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Koji Hattori , Yoichi Munehira , Hideki Kobayashi , Taku Satoh , Shinji Sugiura , Toshiyuki Kanamori

    We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm2 in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 105) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm2. Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm2.





  • Effect of plasma-irradiated silk fibroin in bone regeneration
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Ryoichiro Uchida , Ujjal K. Bhawal , Hideo Kiba , Kiyoshi Arai , Yasuhiro Tanimoto , Noboru Kuboyama , Tetsuo Asakura , Norihiro Nishiyama

    We have recently identified plasma-irradiated silk fibroin (P-AF) as a key regulator of bone matrix properties and composition. Bone matrix properties were tested in 48 femur critical size defects (3.25 mm in diameter) with the expression of osteoblast specific genes at 1 and 2 weeks after surgery. The scaffolds were characterized by various states of techniques; the scanning electronic microcopy revealed the large sized pores in the aqueous-based silk fibroin (A-F) scaffold and showed no alteration into the architecture by the addition of plasma irradiation. The contact angle measurements confirmed the introduction of plasma helped to change the hydrophobic nature into hydrophilic. The histological analyses confirmed the presence of silk fibroin in scaffolds and newly formed bone around the scaffolds. Immunohistochemical examination revealed the increased expression pattern in a set of osteoblast specific genes (TGF-β, TGF-β type III receptor, Runx2, type I collagen and osteocalcin). These data were the first to show that the properties of bone matrix are regulated, specifically through Runx2 pathway in P-AF group. Thus, an employment of P-AF increases several compositional properties of bone, including increased bone matrix, mineral concentration, cortical thickness, and trabecular bone volume.





  • Expression of a novel recombinant fusion protein BVN-Tβ4 and its effects on diabetic wound healing
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Qingfeng Zhou , Shuna Jiang , Kang Ma , Chengwei Li

    A recombinant fusion protein BVN-Tβ4 is successfully expressed in Escherichia coli, purified in the laboratory and applied onto the wounds of diabetic mice to investigate its effects on diabetic wound healing. Our results show that the recombinant protein BVN-Tβ4 can promote diabetic wound healing in the murine model.





  • System-on-fluidics immunoassay device integrating wireless radio-frequency-identification sensor chips
    Publication date: September 2014
    Source:Journal of Bioscience and Bioengineering, Volume 118, Issue 3

    Author(s): Yoshiaki Yazawa , Tadashi Oonishi , Kazuki Watanabe , Akiko Shiratori , Sohei Funaoka , Masao Fukushima

    A simple and sensitive point-of-care-test (POCT) device for chemiluminescence (CL) immunoassay was devised and tested. The device consists of a plastic flow-channel reactor and two wireless-communication sensor chips, namely, a photo-sensor chip and a temperature-sensor chip. In the flow-channel reactor, a target antigen is captured by an antibody immobilized on the inner wall of the flow-channel and detected with enzyme labeled antibody by using CL substrate. The CL signal corresponding to the amount of antigen is measured by a newly developed radio-frequency-identification (RFID) sensor, which enables batteryless operation and wireless data communication with an external reader. As for the POCT device, its usage environment, especially temperature, varies for each measurement. Hence, temperature compensation is a key issue in regard to eliminating dark-signal fluctuation, which is a major factor in deterioration of the precision of the POCT device. A two-stage temperature-compensation scheme was adopted. As for the first stage, the signals of two photodiodes, one with an open window and one with a sealed window, integrated on the photo-sensor chip are differentiated to delete the dark signal. As for the second stage, the differentiated signal fluctuation caused by a temperature variation is compensated by using the other sensor chip (equipped with a temperature sensor). The dark-level fluctuation caused by temperature was reduced from 0.24 to 0.02 pA/°C. The POCT device was evaluated as a CL immunoassay of thyroid-stimulating hormone (TSH). The flow rate of the CL reagent in the flow channel was optimized. As a result, the detection limit of the POCT device was 0.08 ng/ml (i.e., 0.4 μIU/ml).