Characterization of an efficient CRISPR-iCas9 system in Yarrowia lipolytica for the biosynthesis of carotenoids.

The application of clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas9) technology in the genetic modification of Yarrowia lipolytica is challenged by low efficiency and low throughput. Here, a highly efficient CRISPR-iCas9 (with D147Y and P411T mutants) genetic manipulation tool was established for Y. lipolytica, which was further utilized to integrate carotene synthetic key genes and significantly improve the target product yield. First, CRISPR-iCas9 could shorten the time of genetic modification and enable the rapid knockout of nonsense suppressors. iCas9 can lead to more than 98% knockout efficiency for NHEJ-mediated repair after optimal target disruption of a single gene, 100% knockout efficiency for a single gene-guided version, and more than 80% knockout efficiency for multiple genes simultaneously in Y. lipolytica. Subsequently, this technology allowed for rapid one-step integration of large fragments (up to 9902-bp) of genes into chromosomes. Finally, YL-ABTG and YL-ABTG2Z were further constructed through CRISPR-iCas9 integration of key genes in a one-step process, resulting in a maximum ß-carotene and zeaxanthin content of 3.12 mg/g and 2.33 mg/g dry cell weight, respectively. Therefore, CRISPR-iCas9 technology provides a feasible approach to genetic modification for efficient biosynthesis of biological compounds in Y. lipolytica. KEY POINTS: • iCas9 with D147Y and P411T mutants improved the CRISPR efficiency in Y. lipolytica. • CRISPR-iCas9 achieved efficient gene knockout and integration in Y. lipolytica. • CRISPR-iCas9 rapidly modified Y. lipolytica for carotenoid bioproduction.

Developing efficient vanillin biosynthesis system by regulating feruloyl-CoA synthetase and enoyl-CoA hydratase enzymes.

Vanillin is one of the most commonly used natural-occurring flavors in the world. This study successfully constructed an efficient whole-cell catalytic system for vanillin biosynthesis from ferulic acid by regulating feruloyl-CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). First, we constructed an efficient cell-free catalytic system with FCS-Str (fcs from Streptomyces sp. V-1) and ECH-Str (ech from Streptomyces sp. V-1) combination at 1:1. The efficient cell-free catalytic system provided necessary strategies for optimizing the whole-cell catalytic system. Then, we constructed the recombinant Escherichia coli by heterologously expressing the fcs-Str and ech-Str combination. Moreover, E. coli JM109 was a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. Finally, we first adjusted the ratio of FCS and ECH in E. coli JM109 to 1:1 using two copies of fcs-Str. For higher vanillin production, we further optimized the induction conditions of E. coli JM109 to increase the amount of FCS and ECH. The optimized E. coli JM109-FE-F constructed in this study has the highest vanillin synthesis rate of converting 20 mM ferulic acid to 15 mM vanillin in 6 h among all of the E. coli catalytic systems. Our study made a significant contribution to the construction of the vanillin biosynthesis system and provided a valuable strategy for increasing vanillin production. KEY POINTS: • The efficient cell-free vanillin biosynthesis system was constructed by FCS-Str and ECH-Str combination at 1:1. • Escherichia coli JM109 was determined as a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. • Escherichia coli JM109-FE-F with two copies of fcs-Str and one copy of ech-Str has the highest catalytic efficiency for vanillin production.

Enhanced ß-carotene production by overexpressing the DID2 gene, a subunit of ESCRT complex, in engineered Yarrowia lipolytica.

OBJECTIVE: ß-Carotene has been widely used in the food and feed industry and has significant commercial value. This study aimed to increase the ß-carotene production in engineered Yarrowia lipolytica by optimizing the host metabolic network. The DID2 gene, a subunit of the endosomal sorting complex required for transport (ESCRT), was integrated into a ß-carotene producing strain. RESULTS: The ß-carotene production was increased by 260%, and the biomass increased by 10% for engineered Y. lipolytica. Meanwhile, DID2 elevated the mRNA level and protein level of the genes in the ß-carotene synthesis pathway, then increased precursors (FPP, Lycopene) utilization. DID2 also increased the mRNA level of the genes in the glucose pathway, pentose phosphate pathway, and tricarboxylic acid cycle and promoted glucose utilization and cofactors consumption. CONCLUSION: The ESCRT protein complex subunit, DID2, improved ß-carotene production in engineered Y. lipolytica and beneficial to glucose utilization and cofactors consumption. This study provided new finding of the DID2 gene's function and it mostly could be used for many other natural product productions.

Increased campesterol synthesis by improving lipid content in engineered Yarrowia lipolytica.

Sterols attract increasing attention due to their important bioactivities. The oleaginous yeast Yarrowia lipolytica has large lipid droplets, which provide storage for the accumulated steroid compounds. In this study, we have successfully constructed a campesterol biosynthetic pathway by modifying the synthetic pathway of ergosterol in Y. lipolytica with different capacity of lipid synthesis. The results showed that the maximal campesterol production was produced in the engineered strain YL-D+M-E-, as the optimal lipid content. Furthermore, we found that campesterol mainly exists in the lipid droplets. The campesterol production was further accumulated through the overexpression of two copies of dhcr7. Finally, the maximal campesterol production of 837 mg/L was obtained using a 5-L bioreactor in the engineered YL-D+D+M-E-, exhibiting a 3.7-fold increase compared with the initial strain YL-D+E-. Our results demonstrate that the proper promotion of lipid content plays an important role in campesterol biosynthesis in Y. lipolytica, and what we found provides an effective strategy for the production of hydrophobic compounds.Key Points• Campesterol was biosynthesized by deleting erg5 and introducing heterologous dhcr7.• Campesterol production elevated via promotion of lipid content.• Campesterol was mainly found in lipid droplets.• Promotion of lipid content is an effective strategy to produce hydrophobic compounds.

An oncogenic activity of PDGF-C and its splice variant in human breast cancer.

Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer.

Walnut protein isolate based emulsion as a promising delivery system enhanced lutein bioaccessibility.

Lutein, a natural pigment with multiple beneficial bioactivities, faces limitations in food processing due to its instability. In this study, we constructed four modified walnut protein isolate (WNPI) based emulsions as emulsion-based delivery systems (EBDS) for lutein fortification. The modification treatments enhanced the encapsulation efficiency of the WNPI-based EBDS on lutein. The modified WNPI-based EBDS exhibited improved storage and digestive stability, as well as increased lutein delivery capability in simulated gastrointestinal conditions. After in vitro digestion, the lutein retention in the modified WNPI-based EBDS was higher than in the untreated WNPI-based EBDS, with a maximum retention of 49.67 ±â€¯1.10 % achieved after ultrasonic modification. Furthermore, the modified WNPI-based EBDS exhibited an elevated lutein bioaccessibility, reaching a maximum value of 40.49 ±â€¯1.29 % after ultrasonic modification, nearly twice as high as the untreated WNPI-based EBDS. Molecular docking analysis indicated a robust affinity between WNPI and lutein, involving hydrogen bonds and hydrophobic interactions. Collectively, this study broadens WNPI's application and provides a foundation for fortifying other fat-soluble bioactive substances.

Chlorogenic Acid Ameliorates High-Fat and High-Fructose Diet-Induced Cognitive Impairment via Mediating the Microbiota-Gut-Brain Axis.

Chlorogenic acid (CGA) displays cognition-improving properties, but the underlying mechanisms remain unclear. Herein, CGA supplementation (150 mg/kg body weight) for 14 weeks significantly prevented obesity and insulin resistance, cognitive-behavioral disturbances, and synaptic dysfunction induced by a high-fat and high-fructose diet (HFFD). Moreover, CGA supplementation enhanced the expression of genes enriched in the neuroactive ligand-receptor interaction pathway and reduced inflammatory factor expressions. Furthermore, CGA treatment increased gut microbiota diversity and the level of bacterial genera producing SCFAs. CGA also decreased the concentration of energy metabolism substrates, while it increased phosphorylcholine. Finally, we observed a significant correlation among synaptic transmission genes, gut microbiota, and neurotransmission in the CGA supplementation group by targeted multiomics analysis. Together, our results supported that the alteration of gut microbiota and metabolite composition is the underlying mechanism of CGA improving cognitive function. CGA is also a promising intervention strategy to prevent HFFD-induced cognitive impairment.

Resveratrol inhibits lipid and protein co-oxidation in sodium caseinate-walnut oil emulsions by reinforcing oil-water interface.

Lipid-protein co-oxidation often causes nutrition loss, texture changes, and shortened shelf-life of emulsions. In this study, resveratrol significantly prevented lipid-protein co-oxidation in sodium caseinate (NaCas)-walnut oil emulsions, and the underlying mechanisms were explored in physical and chemical aspects. NaCas-walnut oil emulsions stabilized by resveratrol exhibited excellent physical stability at 55 °C for 12 days or at room temperature for 10 months due to forming a stable interfacial layer composed of resveratrol-modified NaCas. Furthermore, resveratrol binding caused NaCas structure's partial unfolding and a âˆ¼ 8% increase in hydrophobicity, in turn enhancing NaCas' emulsification properties and electrostatic repulsion. Besides, more than 90% of resveratrol was loaded at the interface and enhanced NaCas' Fe2+ chelating, DPPH scavenging abilities, and O2 quenching by âˆ¼ 22.6%, 5.26 times, and 31.84%, respectively. Simultaneously, resveratrol significantly improved NaCas' oxidative stability, as reflected by the decrease in adsorbed NaCas' intrinsic fluorescence loss and protein carbonyls gain by âˆ¼ 30% and 37%, respectively. Simultaneously, lipid hydroperoxides and TBARS were reduced by âˆ¼ 30% and 20% in the NaCas-walnut oil emulsions containing 6 mM resveratrol than the control. Our findings contribute to further understanding of the possible interaction among lipid, protein, polyphenols, and their oxidative products at the oil-water interface, minimizing lipid-protein co-oxidation and extending functional oils' shelf life. Finally, walnut oil emulsions with high physical and oxidative stabilities using resveratrol were prepared, further broadening resveratrol's application in the food industry.

Screening low-methanol and high-aroma produced yeasts for cider fermentation by transcriptive characterization.

The commercial active dry yeast strains used for cider production in China are far behind the requirements of the cider industry development in recent decades. In this study, eight yeasts, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia bruneiensis, and Pichia kudriavzevii, were screened and assessed by growth performance, methanol production, aroma analysis, and their transcriptive characterization. Saccharomyces cerevisiae strains WFC-SC-071 and WFC-SC-072 were identified as promising alternatives for cider production. Strains WFC-SC-071 and WFC-SC-072 showed an excellent growth capacity characterized by 91.6 and 88.8% sugar utilization, respectively. Methanol production by both strains was below 200 mg/L. Key aroma compounds imparting cider appreciably characteristic aroma increased in cider fermented by strains WFC-SC-071 and WFC-SC-072. RT-qPCR analysis suggested that most genes associated with growth capacity, carbohydrate uptake, and aroma production were upregulated in WFC-SC-071 and WFC-SC-072. Overall, two Saccharomyces cerevisiae strains are the optimal starters for cider production to enable the diversification of cider, satisfy the differences in consumer demand, and promote cider industry development.

Vitality, fermentation, aroma profile, and digestive tolerance of the newly selected Lactiplantibacillus plantarum and Lacticaseibacillus paracasei in fermented apple juice.

Background: To enrich the probiotic lactic acid bacteria (LAB) strains and expand the commercialization of new fermented juice products, we have identified two LAB strains with excellent potential in fermenting apple juice from pickles. Methods: The two strains were morphologically, physiologically, and genetically characterized. The strains' fermentation performance and alterations in volatile aroma components of apple juice and ability to survive in a simulated gastrointestinal environment were evaluated. Results: Two strains were identified as Lacticaseibacillus paracasei (WFC 414) and Lactiplantibacillus plantarum (WFC 502). The growth of WFC 414 and WFC 502 in apple juice for 48 h reached 8.81 and 9.33 log CFU/mL, respectively. Furthermore, 92% and 95% survival rates were achieved in 2 h simulated gastric juice, and 80.7 and 83.6% survival rates in 4 h simulated intestinal juice. During the fermentation, WFC 414 and WFC 502 reduced the soluble sugars and total polyphenols in apple juice, and consumed malic acid to produce large amounts of lactic acid (3.48 and 5.94 mg/mL). In addition, the esters and aldehydes were reduced, and the production of alcohols, acids and ketones was elevated in the apple juice fermented by both strains. Conclusion: These results show that WFC 414 and WFC 502 have great potential applications in the fermented fruit juice industry.

Structural modification induced by heat treatments improves the emulsifying attributes of lacquer seed protein isolate.

The lacquer seed oil has received extensive attention in the food industry due to its health function, such as regulating blood lipids. But its by-product, lacquer seed meal, is often used as a low-value-added product such as animal feed. Lacquer seed meal contains about 20 % protein, which has amphiphilic properties, and there is limited attention to its emulsifying properties. In this study, the impact of heat treatment on the emulsifying properties of lacquer seed protein isolate (LSPI) was investigated. The EAI and ESI of the 120 °C heated LSPI increased by 77.1 % and 55.2 %, respectively. The emulsions prepared using heat-modified LSPI (120 °C) further showed lower hydroperoxide, TBARS and protein carbonyl contents (only 61.3 %, 61.0 % and 58.6 % of control) after storage. This result indicates that heat-treated LSPI retarded lipid and protein oxidation in LSPI-stabilized emulsions during storage. Changes in protein structure showed that increasing heating temperature resulted in the depolymerization of tertiary structure, higher surface hydrophobicity and lower contents of α-helix of LSPI. These changes in protein structure made the heated LSPIs have better emulsifying properties. Therefore, these findings developed a new use of LSPI and greatly enhanced the potential of LSPI as a natural emulsifier in the food industry.

Elevated ß-Carotene Production Using Codon-Adapted CarRA&B and Metabolic Balance in Engineered Yarrowia lipolytica.

ß-carotene is a precursor of vitamin A and has multiple physiological functions. Producing ß-carotene by microbial fermentation has attracted much attention to consumers' preference for natural products. This study focused on improving ß-carotene production by constructing codon-adapted genes and minimizing intermediate accumulation. The codon-adapted CarRA and CarB genes from the industrial strain of Blakeslea trispora were integrated into the genome of the Yarrowia lipolytica to construct YL-C0, the baseline strain for producing ß-carotene. Thereafter, the ß-carotene biosynthetic pathway's metabolic balance was accurately regulated to reduce the intermediates' accumulation. Notably, the ß-carotene content increased by 21 times to reach 12.5 dry cell weight (DCW) mg/g when minimizing HMG-CoA and FPP accumulation. Further, we improved the expression levels of the CarRA and CarB genes to minimize the accumulation of phytoene and lycopene. Total production of ß-carotene of 1.7 g/L and 21.6 mg/g DCW was achieved. These results reveal that the rate-limiting enzymes CarRA and CarB of B. trispora exhibited higher catalytic activity than the same enzymes from other microorganisms. Promoting metabolic balance by minimizing the accumulation of intermediates is a very effective strategy for increasing ß-carotene. The ß-carotene-producing strain constructed in this study has established the foundation for its potential use in industrial production. These successful engineering strategies also provide a foundation for large-scale production of other terpenoids.

Dissolved-oxygen feedback control fermentation for enhancing ß-carotene in engineered Yarrowia lipolytica.

The DO-stat fed-batch fermentation was carried out to explore the volumetric productivity of ß-carotene in engineered Yarrowia lipolytica C11 strain. Using DO-stat fed-batch fermentation, we achieved 94 g/L biomass and 2.01 g/L ß-carotene. Both biomass and ß-carotene were about 1.28-fold higher than that in fed-batch fermentation. The ATP, NADP+/NADPH, and gene expression levels of tHMG, GGS1, carRA, and carB were promoted as compared to that in fed-batch fermentation. As for as the kinetic parameters in DO-stat fed-batch fermentation, µm', Yx/s', and Yp/s' was 0.527, 0.353, and 0.158, respectively. The µm' was elevated 4.66-fold than that in fed-batch fermentation. These data illustrate that more dissolved oxygen increased the biomass. The Yx/s' and Yp/s' were increased 1.15 and 22.57-fold, which suggest that the DO-stat fed-batch fermentation reduced the Crabtree effect and improved the utilization rate of glucose. Therefore, DO-stat fed-batch fermentation is a promising strategy in the industrialized production of ß-carotene.

Overexpression of △12, △15-Desaturases for Enhanced Lipids Synthesis in Yarrowia lipolytica.

Microbial oil triacylglycerol (TAG) from the renewable feedstock attract much attention. The oleaginous yeast Yarrowia lipolytica has become the most studied for lipid biosynthesis. Fatty acid desaturases catalyze the introduction of a double bond into fatty-acid hydrocarbon chains to produce unsaturated fatty acids. Desaturases are known to enhance lipid accumulation. In this study, we have achieved a significant increase in lipid production and increase the unsaturated fatty acids content in Y. lipolytica. By comparing the expression of the native genes of △-9 stearoyl-CoA desaturase (SCD) and △12 desaturase (△12D), and an exogenous △15 desaturase (△15D) from flax in the strain with deleted peroxisomal biogenesis factor 10 (PEX10) and overexpressed diacylglyceride acyl-transferase (DGA1), we found that the strain with overexpressed △15 desaturase accumulated 30.7% lipid. Simultaneously, we explored the effect of two copies of desaturase genes (12D-SCD, 15D-SCD, 12D-15D) on lipid production, and found co-expression of △12D and △15D accumulated 42.6% lipid. The lipid content was further increased by 56.3% through the deletion of the multifunctional enzyme (MFE1) and the overexpression of acetyl-CoA carboxylase (ACC1). Finally, the lipid productivity of 50 g/L and maximal lipid content of 77.8% DCW are obtained using a 5-L stirred-tank bioreactor during the stationary phase in the engineered YL-10. Our result demonstrated that the △12 and △15 desaturases play an important role in lipid production in Y. lipolytica and provides an effective strategy for biodiesel development.

Promoting the Synthesis of Precursor Substances by Overexpressing Hexokinase (Hxk) and Hydroxymethylglutaryl-CoA Synthase (Erg13) to Elevate ß-Carotene Production in Engineered Yarrowia lipolytica.

As a valuable carotenoid, ß-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of ß-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and ß-carotene biosynthesis, this study aims to increase the ß-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered Yarrowia lipolytica. In this study, we investigated the effect of the unique hexokinase gene (Hxk) overexpression on ß-carotene accumulation and glucose consumption. The Hxk gene was introduced into a ß-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the ß-carotene content by 98%. Overexpression of the Hxk gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of Hxk (315% higher) compared to the control Y.L-1 strain. Moreover, Hxk overexpression accelerated the utilization rate of glucose. The gene erg13 encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for ß-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of erg13 produced 8.41 mg/g dry cell weight (DCW) of ß-carotene, which was 259% higher than Y.L-1. The ß-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating erg13 into the chromosome and Hxk overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the erg13 gene. Finally, the titer of ß-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of Hxk and erg13 could improve ß-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of ß-carotene synthesis.

Phage M13KO7 detection with biosensor based on imaging ellipsometry and AFM microscopic confirmation.

A rapid detection and identification of pathogens is important for minimizing transfer and spread of disease. A label-free and multiplex biosensor based on imaging ellipsometry (BIE) had been developed for the detection of phage M13KO7. The surface of silicon wafer is modified with aldehyde, and proteins can be patterned homogeneously and simultaneously on the surface of silicon wafer in an array format by a microfluidic system. Avidin is immobilized on the surface for biotin-anti-M13 immobilization by means of interaction between avidin and biotin, which will serve as ligand against phage M13KO7. Phages M13KO7 are specifically captured by the ligand when phage M13KO7 solution passes over the surface, resulting in a significant increase of mass surface concentration of the anti-M13 binding phage M13KO7 layer, which could be detected by imaging ellipsometry with a sensitivity of 10(9)pfu/ml. Moreover, atomic force microscopy is also used to confirm the fact that phage M13KO7 has been directly captured by ligands on the surface. It indicates that BIE is competent for direct detection of phage M13KO7 and has potential in the field of virus detection.

Soluble angiopoietin receptor Tie-2 in patients with acute myocardial infarction and its detection by optical protein-chip.

The Tie-2 receptor has been shown to play a role in angiogenesis in atherosclerosis. The conventional method assaying the level of soluble Tie-2 (sTie-2) was ELISA. However, this method has some disadvantages. The aims of this research are to establish a more simple detection method, the optical protein-chip based on imaging ellipsomtry (OPC-IE) applying to Tie-2 assay. The sTie-2 biosensor surface on silicon wafer was prepared first, and then serum levels of sTie-2 in 38 patients with AMI were measured on admission (day 1), day 2, day 3 and day 7 after onset of chest pain and 41 healthy controls by ELISA and OPC-IE in parallel. Median level of sTie-2 increased significantly in the AMI patients when compared with the controls. Statistics showed there was a significant correlation in sTie-2 results between the two methods (r=0.923, P<0.01). The result of this study showed that the level of sTie-2 increased in AMI, and OPC-IE assay was a fast, reliable, and convenient technique to measure sTie-2 in serum.

Elevated ß-Carotene Synthesis by the Engineered Rhodobacter sphaeroides with Enhanced CrtY Expression.

ß-Carotene is a precursor of vitamin A and a dietary supplement for its antioxidant property. Producing ß-carotene by microbial fermentation has attracted much attention owing to consumers' preference for the natural product. In this study, an engineered photosynthetic Rhodobacter sphaeroides producing ß-carotene was constructed by the following strategies: (1) five promoters of different strengths were used to investigate the effect of the expression level of crtY on ß-carotene content. It was found that PrrnB increased the ß-carotene content by 109%. (2) blocking of the branched pentose phosphate pathway by zwf deletion, and (3) overexpressing dxs could restore the transcriptional levels of crtE and crtB. Finally, the engineered RS-C3 has the highest ß-carotene content of 14.93 mg/g dry cell weight (DCW) among all of the reported photosynthetic bacteria and the ß-carotene content reached 3.34 mg/g DCW under light conditions. Our results will be available for industrial use to supply a large quantity of natural ß-carotene.

A label-free protein microfluidic array for parallel immunoassays.

A label-free protein microfluidic array for immunoassays based on the combination of imaging ellipsometry and an integrated microfluidic system is presented. Proteins can be patterned homogeneously on substrate in array format by the microfluidic system simultaneously. After preparation, the protein array can be packed in the microfluidic system which is full of buffer so that proteins are not exposed to denaturing conditions. With simple microfluidic channel junction, the protein microfluidic array can be used in serial or parallel format to analyze single or multiple samples simultaneously. Imaging ellipsometry is used for the protein array reading with a label-free format. The biological and medical applications of the label-free protein microfluidic array are demonstrated by screening for antibody-antigen interactions, measuring the concentration of the protein solution and detecting five markers of hepatitis B.

[Application of optical proteinchip in detecting phage M13KO7].

Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.