A Whole-Cell Surface Plasmon Resonance Sensor Based on a

Feb 1, 2016 - ... Displaying a Gold-Binding Protein: Usefulness for Diagnosis of Maple Syrup Urine Disease ... different statuses of maple syrup urine...
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A whole-cell surface plasmon resonance sensor based on a leucine auxotroph of Escherichia coli displaying a gold-binding protein: usefulness for diagnosis of maple syrup urine disease Min-Ah Woo, Jung Hun Park, Dae-Yeon Cho, Sang Jun Sim, Moon Il Kim, and Hyun Gyu Park Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04648 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 1, 2016

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A whole-cell surface plasmon resonance sensor based on a leucine auxotroph of Escherichia coli displaying a goldbinding protein: usefulness for diagnosis of maple syrup urine disease

Min-Ah Woo,†,‡ Jung Hun Park,† Daeyeon Cho,# Sang Jun Sim,Ⅱ Moon Il Kim,§* and Hyun Gyu Park†*



Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro,

Yuseong-gu, Daejeon 34141, Republic of Korea ‡

Food Safety Research Group, Korea Food Research Institute, Baekhyun-dong, Bundang-gu, Seongnam-si,

Gyeonggi-do 463-746, Republic of Korea #

LabGenomics Co., Ltd., 1571-17 Seocho3-dong, Seocho-gu, Seoul 137-874, Republic of Korea



Department of Chemical and Biological Engineering, Korea University, Anam-Dong 5-1,

Seongbuk-Gu, Seoul 136-713, Republic of Korea §

Department of BioNano Technology, Gachon University, 1342 Seongnamdae-ro, Sujeong-gu, Seongnam-si,

Gyeonggi-do 461-701, Republic of Korea

Correspondence: Hyun Gyu Park, Ph.D., Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea, Tel.: +82 42-350-3932, Fax: +82 42-350-3910, E-mail: [email protected]

Moon Il Kim, Ph.D., Department of BioNano Technology, Gachon University, 1342 Seongnamdae-ro, Sujeong-gu, Seongnam-si, Gyeonggi-do 461-701, Republic of Korea, Tel: +82 31-750-8563, Fax: +82 31750-8774, E-mail: [email protected] 1

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Abstract We developed a whole-cell surface plasmon resonance (SPR) sensor based on a leucine auxotroph of Escherichia coli displaying a gold-binding protein (GBP) in response to cell growth and applied this sensor to diagnosis of maple syrup urine disease, which is represented by the elevated leucine level in a blood. The leucine auxotroph was genetically engineered to grow displaying GBP in a proportion to the concentration of target amino acid leucine. The GBP expressed on the surface of the auxotrophs directly bound to the golden surface of an SPR chip without the need for any additional treatment or reagents, which consequently produced SPR signals used to determine leucine levels in a test sample. Gold nanoparticles (GNPs) were further applied to the SPR system, which significantly enhanced the signal intensity up to 10-fold by specifically binding to GBP expressed on the cell surface. Finally, the diagnostic utility of our system was demonstrated by its employment in reliably determining different status of maple syrup urine disease based on a known cutoff level of leucine. This new approach based on an amino acidauxotrophic E. coli strain expressing a GBP that binds to an SPR sensor, holds great promise for detection of other metabolic diseases of newborn babies including homocystinuria and phenylketonuria, which are also associated with abnormal levels of amino acids.

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Introduction The goal of clinical testing of newborns is the diagnosis of a list of congenital diseases that can be fatal unless they are properly treated at an early stage.1,2 Such diseases include metabolic disorders and blood- or hormone-related problems2, however, most of them are metabolic disorders. The latter diseases, often called “inborn errors of metabolism,” are caused by the lack of specific enzymes that are involved in the body’s utilization of nutrients for maintenance of healthy tissues and for the production of energy.3 As a consequence of metabolic disorders, an infant’s normal development can be seriously hindered; for example, fatal damages can occur during the development of organs such as the cerebrum, eyeballs, and kidneys.4 Therefore, the screening of newborns is highly important and essential for predicting and preventing those damages, and is currently mandatory in many countries.5,6 To date, several methods have been developed for the screening of newborns to diagnose frequently occurring metabolic disorders such as congenital hypothyroidism, phenylketonuria (PKU), maple syrup urine disease, homocystinuria, galactosemia, and congenital adrenal hyperplasia. In 1961, scientist R. Guthrie developed a bacterial inhibition assay (BIA) that could determine whether newborns had a metabolic disorder, PKU, via analysis of blood samples collected on a specially designed filter.7 Before long, this method was also utilized for detection of other metabolic diseases such as galactosemia, maple syrup urine disease, and homocystinuria. In 1992, tandem mass spectrometry-based diagnosis of metabolic disorders was introduced. This method can be used to test newborns for more than 40 kinds of inherited metabolic diseases simultaneously using a single drop of blood8; however, this method required laborious pre- and post-treatment steps as well as expensive instrumentation. Since the enzymatic colorimetric assay was developed in 1996, it has been the most commonly used method because it is relatively simple and accurate. More recently, an enzymatic method for screening of newborns for 20 types of lysosomal storage diseases was developed.9 3

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Each existing method has its own advantages and disadvantages. After the enzymatic colorimetric assay was developed, the Guthrie test has been hardly used because of the risk of inaccurate results and because it is a passive test system. As described above, tandem mass spectrometry involves high costs and requires complex prehandling of blood samples and a highly skilled operator. An enzymatic colorimetric assay is faster and simpler than tandem mass spectrometry but requires special equipment and many reagents for a number of enzymatic reactions.9 SPR has been frequently used for real-time analysis of specific bimolecular interactions involving DNA, proteins, or cells without labeled signaling probes such as fluorescent agents.10 The detection is based on the SPR phenomenon that occurs when light is reflected off thin metal films. If there are specific interactions of molecules that are attached or immobilized on a metal surface, the refractive index of the adjacent medium changes with high sensitivity.11 Since it is possible to quantitatively analyze target molecules by monitoring the change in the refractive index, SPR has been extensively employed in diverse biosensing applications.12,13 Recently, gold-binding protein (GBP) was utilized to achieve an advanced SPR-based biosensing systems taking advantage of its specific interaction with the golden surface of a SPR chip.14-17 Along this line, we utilized a GBP-displaying auxotroph to achieve a whole-cell SPR sensor for diagnosis of maple syrup urine disease, which is represented by the elevated leucine level in a blood as a major metabolic disorder among newborns. A leucine auxotroph was genetically engineered to grow only in the presence of leucine; thus, its growth was proportional to the leucine concentration in a test sample. The pTacFadLGBP-1 plasmid, which expresses a GBP on the cell surface as the cells grow, was introduced into the leucine auxotroph, so that the growing/proliferating cells simply and directly bind to the gold surface of an SPR chip without any additional treatment or reagents. Gold nanoparticles (GNPs), which are capable of specifically binding to a GBP on the cell surface, were further applied to the SPR chip after the binding of the cells to enhance the SPR signal up to

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10-fold. Based on the SPR signals induced by the presence of leucine, we reliably diagnosed different status of maple syrup urine disease using real clinical samples.

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Experimental Section Construction of a Leucine Auxotroph Displaying a GBP In our previous report,18 a leucine auxotroph was constructed via repetitive selection from a library produced by transposon mutagenesis. Briefly, 1 µL of the KAN-2 transposon (Epicenter, Wisconsin, USA) was electroporated into the Escherichia coli W strain (ATCC, Manassas, Virginia, USA; cat. # ATCC1105)19 by using standard procedures.20 Transposon insertion mutants were selected on a Luria-Bertani (LB) agar plate containing kanamycin (Km; 50 µg/mL). Among the transposon-5 (Tn5) insertional mutants, leucine auxotrophs were selected by means of replica plates, first on an M9 medium plate containing Km and leucine and then on an M9 medium plate containing Km without leucine. After selection of mutants that were likely leucine auxotrophs by means of the above-mentioned plate selection, we confirmed the auxotrophic characteristics of candidates in 3 mL of the M9 liquid medium with or without leucine. To display GBP on the cell surface, we used the previously designed pTacFadLGBP-1 plasmid (Apr, pTac99A derivative, fadL, GBP, 6.9 kb).21 The leucine auxotroph served as a host strain for the cell surface display of the FadL–GBP fusion protein. Competent cells that originated from the auxotroph were first prepared by washing the cells with ice-cold water three times. Then the pTacFadLGBP-1 plasmid was electroporated into the competent cells on a Gene Pulser system (Bio-Rad, California, USA). The cells were then grown in LB agar plates containing ampicillin (Am; 50 µg/mL) at 37°C overnight. After that, the colonies were collected and grown in LB broth containing Am (50 µg/mL) at 37°C for 12 h. The cell suspension was stored in 20% glycerol at −70°C.

Membrane Fraction Analysis The leucine auxotroph transformant was cultivated in LB broth containing 50 µg/mL each of Am and Km at 37°C for 9–12 h with shaking. After the cells were washed with the M9 medium twice, GBP was expressed by cultivating 6 × 109 cells at 37°C for 6 h in a shaking incubator at 200 rpm in 6

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6 mL M9 media including 10 nM cyanocobalamin (Sigma-Aldrich, St. Louis, Missouri, USA), 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, Missouri, USA), and 50 µM leucine. To isolate outer-membrane proteins, 6 mL of the culture broth was centrifuged at 4,000 rpm at 4°C for 5 min, and the cell pellet was washed with 1 mL of 10 mM Na2HPO4 buffer (pH 7.3) twice; this action was followed by centrifugation at 4,000 rpm at 4°C for 5 min. The resulting cell pellet was resuspended in 400 µL of 10 mM Na2HPO4 buffer (pH 7.3) containing 5 mM MgCl2, 5 mM β-mercaptoethanol, 2 mg/mL lysozyme, 10 µg/mL RNase A, and 5 µg/mL DNase I. Crude extracts of the cells were prepared by means of three cycles of [i] sonication (in a sonicator at temperature maintained below 10°C for 5 min), [ii] freezing (in liquid nitrogen for 5 min), and [iii] thawing (in a water bath at 37°C for 2 min). Then the solution was centrifuged at 13,000 rpm at 4°C for 20 min, and the pellet was resuspended in 300 µL of 50 mM Tris-HCl (pH 7.3) containing 0.2 M KCl, 5 mM EDTA, and 5 mM β-mercaptoethanol. After separation of the pellet at 13,000 rpm for 10 min at 4°C, the pellet was resuspended in 50 µL of 50 mM Tris-EDTA (pH 8.0). The GBP in the membrane fraction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; in a 12% [w/v] gel).

Sample Preparation The leucine auxotroph was cultivated in the M9 medium containing 50 µg/mL each of Am and Km, 500 µM leucine, and 10 nM cyanocobalamin at 37°C for 9–12 h with shaking. After the cells were washed with the M9 medium twice, 107 cells were resuspended in 3 mL assay solution in the M9 medium containing 10 nM cyanocobalamin, 1 mM IPTG, and 100 µL of a test sample; then the cells were cultivated at 37°C for 6 h in a shaking incubator at 200 rpm to induce GBP expression from the tac promoter. To mimic real blood, amino acid cocktail (described below) was additionally included in the assay solution. All amino acids that were used to prepare the amino acid cocktail were purchased from Sigma-Aldrich, and the cocktail consisted of 300 µM alanine, 40 µM arginine, 7

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60 µM asparagine, 3 µM aspartate, 45 µM cysteine, 500 µM glutamine, 50 µM glutamate, 220 µM glycine, 65 µM histidine, 65 µM isoleucine, 100 µM leucine, 180 µM lysine, 20 µM methionine, 50 µM phenylalanine, 100 µM serine, 120 µM threonine, 60 µM tyrosine, 20 µM tryptophan, 220 µM valine, and 200 µM proline. Cell numbers were counted by measuring absorbance at 600 nm (DU 650 Spectrophotometer, Beckman, Fullerton, California, USA). Dried blood samples of newborns were obtained from LabGenomics (Seoul, Korea). We punched the filter paper specimens (3 mm in diameter) including unknown real clinical blood samples. To elute leucine from the filter paper, the punched paper in a plastic envelope was incubated with 3% trichloroacetic acid (Sigma-Aldrich) for 1 h at room temperature with moderate shaking. After that, 100 µL of the eluted solution was used to prepare the final assay solution. The pH level of the assay solution was adjusted to 7.3 using 200 µM NaHCO3 (Sigma-Aldrich). The unknown leucine concentrations of the test samples were determined on a mass spectrometer (Hitachi 7180, Hitachi, Japan) according to the manufacturer’s instructions and protocols.

SPR Analysis Before the SPR experiments, a sensor chip was dipped into the piranha solution (a 3:1 mixture of concentrated H2SO4 and 30% [v/v] H2O2) for 20 min and then washed with distilled deionized water (DDW). After flow-drying of the SPR sensor chip by using nitrogen gas, DDW was run across the sensor chip surface for 10 min to equilibrate the chip. Next, 200 µL of an assay solution containing the grown cells was loaded at the flow rate of 20 µL/min. After that, 200 µL of DDW was run through to wash out unbound cells, and 200 µL of a 15 nM suspension of GNPs was then applied into the channel to enhance the SPR signals. The unbound GNPs were then washed out with 200 µL of DDW. During each step, including sample loading, washing, and GNP loading, the SPR signals were monitored in fixed-angle mode with 2 µL of flow cell volume on a Mico SPR nano device (MicoBioMed Co., Ltd., Korea) equipped with a desktop computer for SPR data acquisition. 8

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Results and Discussion Whole-Cell SPR Sensor Based on a GBP-Displaying Leucine Auxotroph One of the key points of this study is successful preparation of a leucine auxotroph of E. coli, whose growth is possible only in the presence of leucine and is proportional to the leucine concentration. In our previous study,18 we constructed leucine auxotroph and demonstrated linearity of its growth with leucine by measuring luminescent signals produced by the cell growth. As shown in Figure 1, the pTacFadLGBP-1 plasmid was inserted into the leucine auxotroph, and the gene expression was induced from the tac promoter in the presence of leucine and IPTG to display FadL–GBP fusion proteins on the cell surface. The leucine auxotroph would then bind to the SPR chip through the displayed GBP without the requirement of any external substrate. To further enhance the SPR signal intensity, we applied GNPs, which can directly bind to GBP on the cell surface. Using this method, leucine levels in the test samples could be determined by monitoring the SPR signals produced by cell attachment on the chip and subsequent binding of GNPs to the cells. We first performed membrane fraction analysis of GBP-displaying leucine auxotroph to confirm that GBP was displayed on the cell surface. The cell transformant harboring the pTacFadLGBP-1 plasmid, was cultivated in the M9 medium containing 10 nM cyanocobalamin, 1 mM IPTG, and 50 µM leucine, from which total membrane proteins were extracted. SDS-PAGE analysis of the membrane proteins was then carried out on a 12% gel. As a result, the membrane fraction extracted from the IPTG-induced cells showed a thick 46-kDa band corresponding to FadL–GBP (cells not induced with IPTG did not yield such a band; Supporting Information, Figure S-1). The goldbinding event of the leucine auxotroph displaying the GBP was also examined by running the cell suspension into an SPR channel and measuring SPR signals produced by the cell attachment. As shown in Figure S-2 (Supporting Information), we observed proportional increase of R.U. values (327, 677, and 1,114) according to the increase in cell amounts when three cell suspensions (3 × 105, 9

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3 × 107, and 3 × 109 cells per 200 µL) were passed across the SPR chip surface, respectively.

Leucine-Induced Generation and GNP-Utilized Enhancement of SPR Signals To examine the effects of leucine concentration on the induced SPR signal intensity, leucine auxotroph harboring pTacFadLGBP-1 was cultivated in the M9 medium containing different leucine concentrations (0, 5, and 10 µM), and the cultured cell suspensions were flowed into each channel of the SPR chip. The R.U. values were then measured after washing out the unbound cells with DDW. Figure S-3 (Supporting Information) shows SPR profiles of resonance changes induced by the different leucine concentrations. The resulting R.U. values of the three samples increased proportionally to the leucine concentration: 590, 639, and 721 R.U., respectively. There was some background signal observed from the control sample without leucine due to the nonspecific binding of the initially applied cells to the gold surface of SPR chip. But the samples containing leucine were distinguishable from the control sample although the signal differences between the samples were not large enough to achieve reliable detection of leucine. To enhance the leucine-induced SPR signal intensity, we further applied GNPs to the IPTGinduced cells displaying the GBP which were already attached on the chip. As a result presented in Figure 2, the SPR signals from the IPTG-induced cells displaying GBP significantly increased upon the application of GNPs while there was no GNP-induced signal enhancement observed for the non-induced cells. This result demonstrates that GNPs were successfully attached to the GBP on the cells and led to signal enhancement, which was also confirmed by TEM analyses (Supporting Information, Figure S-4 and S-5). Most importantly, SPR signals from GBPdisplaying cells are quite higher than those from negative cells without GBP, which would lead to the reliable detection of leucine. When we tested three concentrations of GNPs, the SPR signals increased proportionally to the GNP concentration (15 nM GNPs: 9,836 R.U., 10 nM GNPs: 5,768 R.U., and 5 nM GNPs: 2,664 R.U.). Therefore, 15 nM GNPs were used in the further study.

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Diagnostic Utility of the Whole-Cell SPR Sensor for Maple Syrup Urine Disease Maple syrup urine disease is one of the major metabolic disorders among newborns and is caused by a deficiency in the branched-chain α-keto acid dehydrogenase complex, leading to a buildup of branched-chain amino acids (leucine, isoleucine, and valine) and their toxic metabolites in blood and urine.22,23 Newborn babies with this disease seem healthy at birth, but if left untreated, they suffer severe brain damage and eventually die. Therefore, early diagnosis of this disease by measuring the corresponding amino acid concentrations in blood is highly important. A leucine concentration higher than 300 µM in a newborn baby’s blood is generally recognized to be an indicator of maple syrup urine disease23; thus, in our whole-cell SPR sensor system, an R.U. value produced by the attached cells that were grown with 10 µM leucine (1/30 dilution of leucine in the culture medium) was assumed to be the cutoff value. To evaluate the utility of our system for diagnosis of maple syrup urine disease, we first analyzed two artificial samples representing the disease (13 µM leucine) and normal (6 µM leucine) situations (Figure 3). In the presence of leucine representing the disease, cutoff, and normal samples, the GBP-displaying leucine auxotrophs were grown in the M9 media (Figure 3A) or amino acid cocktail mimicking real blood (Figure 3B), which were then subjected to the SPR sensing system. As a result shown in Figure 3, the SPR signals from the disease and normal samples were correctly higher and lower, respectively, than the cutoff signal, confirming the capability of this system to discriminate each clinical status of maple syrup urine disease through the comparison of the resulting SPR signals with a known cutoff signal. Finally, the diagnostic utility of our system was demonstrated by determining the clinical status of real clinical samples extracted from newborn blood filter paper (Figure 4). Three different sets consisting of two normal samples, two disease samples, or one normal and one disease samples were applied to the leucine auxotrophs with the samples representing the cutoff status in maple syrup urine disease and their SPR signals were obtained by introducing the cells into the SPR sensor. The actual leucine concentrations in the test samples were determined by using a mass spectrometer.

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As a result, both the disease and normal samples were very successfully identified in all cases by yielding the respective higher and lower SPR signals than the signals from the cutoff sample.

Conclusions This study shows that a GBP-displaying leucine auxotroph can be successfully used for the development of a whole-cell SPR sensor for diagnosis of maple syrup urine disease. The leucine auxotroph grows in the presence of leucine in a sample and expresses a GBP on the cell surface. Consequently, the GBP-displaying cells simply and directly bind to the gold surface of the SPR chip without any pretreatment or addition of reagents. The cells that are bound to the gold surface yield different SPR signals that are proportional to the leucine concentrations, and the signals are greatly enhanced by applying GNPs. Finally, we were able to successfully diagnose maple syrup urine disease for real blood samples based on known cutoff signal. Our whole-cell SPR sensor system would hold great promise as a simple and convenient diagnostic tool for screening of newborns.

Acknowledgment This work was financially supported by the Center for BioNano Health-Guard funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea as a Global Frontier Project (Grant H-GUARD-2013M3A6B2078964) and by Basic Science Research Program through the NRF funded by the Ministry of Education [No. 2015R1A2A1A01005393]. This study was also supported by the National Research Foundation of Korea (NRF) grants [No. NRF-2013R1A2A1A01015644] of the MSIP of Korea. This research was also supported by the Korea Food Research Institute.

Supporting Information SDS-PAGE analysis of membrane fractions of a leucine auxotroph harboring the pTacFadLGBP-1 plasmid (Figure S-1), SPR profiles induced by the different amounts of leucine auxotrophic cell (Figure S-2) and leucine (Figure S-3), TEM image and UV-vis absorption spectrum of GNPs (Figure S-4), and TEM images showing the interaction between GNPs and leucine auxotrophs (Figure S-5). The Supporting Information is available free of charge on the ACS Publications 12

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website.

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Anal. Biochem. 2002, 302, 28–37. (14) Soh, N.; Tokuda, T.; Watanabe, T.; Mishima, K.; Imato, T.; Masadome, T.; Asano, Y.; Okutani, S.; Niwa, O.; Brown, S. Talanta 2003, 60, 733-745. (15) Park, T. J.; Hyun, M. S.; Lee, H. J.; Lee, S. Y.; Ko, S. Talanta 2009, 79, 295–301. (16) Ko, S.; Park, T. J.; Kim, H.; Kim, J.; Cho, Y. Biosens. Bioelectron. 2009, 24, 2592–2597. (17) Zheng, S., Kim, D., Park, T. J., Lee, S. J., and Lee, S. Y. Talanta 2010, 82, 803–809. (18) Kim, M. I.; Yu, B. J.; Woo, M. A.; Cho, D.; Dordick, J. S.; Cho, J. H.; Choi, B. O.; Park, H. G. Anal. Chem. 2010, 82, 4072-4077. (19) Burkholder, P. R. Science 1951, 114, 459–460. (20) Reznikoff, W. S.; Goryshin, I. Y.; Jendrisak, J. J. J. Methods Mol. Biol.2004, 260, 83–96. (21) Park, T. J.; Zheng, S.; Kang, Y. J.; Lee, S. Y. FEMS Microbiol Lett 2009, 293, 141-147. (22) Park, H. D.; Lee, D. H.; Hong, Y. H.; Kang, D. H.; Lee, Y. K.; Song, J.; Lee, S. Y.; Kim, J. W.; Ki, C. S.; Lee, Y. W. Ann Clin Lab Sci. 2011, 41, 167-73. (23) Janečková, H.; Hron, K.; Wojtowicz, P.; Hlídková, E.; Barešová, A.; Friedecký, D.; Zídková, L.; Hornik, P.; Behúlová, D.; Procházková, D.; Vinohradská, H.; Pešková, K.; Bruheim, P.; Smolka, V.; Sťastná, S.; Adam, T. J Chromatogr A. 2011, 24, 11-17.

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Figure captions

Figure 1. The scheme of the whole-cell SPR sensor by utilizing a GBP-displaying leucine auxotroph.

Figure 2. GNP-mediated enhancement of SPR signals. SPR profiles were obtained via flow of a leucine auxotroph that was not induced with IPTG (a) and of a leucine auxotroph displaying GBP on the surface, which was induced by adding IPTG (b, c, d). The same number of cells (108) was introduced to each channel, and then GNPs at different concentrations—(b) 5 nM, (c) 10 nM, and (d) 15 nM—were further applied. The resulting R.U. values after washing out the unbound GNPs were 2,664, 5,768, and 9,836, respectively.

Figure 3. Diagnosis of maple syrup urine disease for artificial samples. (A) Determination of (a) disease state (13 µM leucine) and (c) normal state (6 µM leucine) of artificial samples in M9 media based on the cut-off value (10 µM leucine, (b)). The resulting R.U. values of the disease, cutoff, and normal samples were 10,027, 8,259, and 6,362, respectively. (B) Determination of (a) disease state (13 µM leucine) and (c) normal state (6 µM leucine) of artificial samples in amino acid cocktail based on the cut-off value (10 µM leucine, (b)). The resulting R.U. values of the disease, cutoff, and normal samples were 16,210, 12,386, and 6,983, respectively.

Figure 4. Diagnosis of maple syrup urine disease for real clinical newborn babies’ blood samples. (A) Two samples representing normal state (8.37 and 6.99 µM leucine), (B) two samples representing disease state (13.35 and 12.16 µM leucine), and (C) two samples representing one normal (5.56 µM) and one disease (16.74 µM) states.

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Table of Contents (TOC)

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