Multiplexed Amino Acid Array Utilizing Bioluminescent Escherichia

Apr 20, 2010 - Min-Ah Woo , Jung Hun Park , Daeyeon Cho , Sang Jun Sim , Moon Il ... Moon Il Kim , Tae Jung Park , Nam Su Heo , Min-Ah Woo , Daeyeon ...
0 downloads 0 Views 3MB Size
Download PDF
Anal. Chem. 2010, 82, 4072–4077

Multiplexed Amino Acid Array Utilizing Bioluminescent Escherichia coli Auxotrophs Moon Il Kim,† Byung Jo Yu,‡ Min-Ah Woo,† Daeyeon Cho,§ Jonathan S. Dordick,| June Hyoung Cho,‡ Byung-Ok Choi,⊥ and Hyun Gyu Park*,† Department of Chemical and Biomolecular Engineering, KAIST 335 Gwahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea, MD Science Inc., 258-1 Munji-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea, LabGenomics Co., Ltd., #1571-17 Seocho3-dong, Seocho-gu, Seoul 137-874, Republic of Korea, Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, and Department of Neurology, College of Medicine Ewha Womans University, Mokdong Hospital, 911-1 Mokdong, Yangcheon-gu, Seoul 158-710, Republic of Korea We describe a novel multiplex “amino acid array” for simultaneously quantifying different amino acids based on the rapid growth of amino acid auxotrophic E. coli. First, we constructed genetically engineered amino acid auxotrophs of E. coli containing a bioluminescence reporter gene, yielding concomitant luminescence as a response to cell growth, and then immobilized the reporter cells within individual agarose of respective wells in a 96-well plate serving as a mimic of a biochip. Using the amino acid array, we were able to determine quantitatively the concentrations of 16 amino acids in biological fluid by simply measuring bioluminescent signals from the immobilized cells within 4 h without pre- and posttreatment. The clinical utility of this method was verified by quantifying different amino acids in dried blood spot specimens from clinical samples for the diagnosis of metabolic diseases of newborn babies. This method serves as a convenient route to the rapid and simultaneous analysis of multiple amino acids from complex biological fluids and represents a new analytical paradigm that can replace conventional, yet laborious methods currently in use. Determining amino acid concentrations is of broad interest in numerous aspects of biological research, clinical diagnosis and medicine, food technology, etc. In particular, measurement of free amino acid concentrations in physiological fluids such as blood, cerebrospinal fluid (CSF), and urine has been used as biochemical indicators of newborn errors of metabolism (aminoacidopathies), nutritional status, and therapeutic monitoring.1-4 * To whom correspondence should be addressed. E-mail: [email protected]. † KAIST 335 Gwahak-ro. ‡ MD Science Inc. § LabGenomics Co., Ltd. | Rensselaer Polytechnic Institute. ⊥ Mokdong Hospital. (1) Scriver, C. R.; Clow, C. L.; Lamm, P. Am. J. Clin. Nutr. 1971, 24, 876–890. (2) Massey, K. A.; Blakeslee, C. H.; Pitkow, H. S. Amino Acids 1997, 14, 271– 300. (3) Hanley, W. B.; Lee, A. W.; Hanley, A. J. G.; Lahotay, D. C.; Austin, V. J.; Schoonheyt, W. E.; Platt, B. A.; Clarke, J. T. R. Mol. Genet. Metab. 2000, 69, 286–294. (4) Elango, R.; Ball, R. O.; Pencharz, P. B. Amino Acids 2009, 37, 19–27.

4072

Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

A number of assays to determine amino acid concentrations in biological fluids have been developed and used including ionexchange chromatography in combination with postcolumn ninhydrin detection, gas chromatography, reversed-phase HPLC after precolumn derivatization, thin-layer chromatography, fluorometric methods, and mass or tandem mass spectrometry.5-11 These assays show little cross-reactivity among amino acids; however, amino acid degradation by common sample pretreatment methods, e.g., using strong acids, organic solvents, ultrafiltration, or anticoagulants, has frequently resulted in poor quantitative analysis.12 Moreover, the intrinsic instability of the ninhydrin reagent for amino acid derivatization often makes this analysis slow and inaccurate. Finally, these methods either involve numerous experimental steps or require complicated and expensive instrumentation for analysis. Thus, there is significant incentive to develop a new approach to enable more convenient, reliable, and economical detection of amino acid concentrations. Along these lines, cell-based assays have been used to quantify amino acids.13 Representatively, auxotrophic mutants of E. coli have been employed to measure the lysine content in test samples.14-16 This approach, however, requires over 10 h to perform. Furthermore, the lack of multiple analyzing power of the approach highly restricts its widespread application clinically. To overcome these limitations, we have developed a solidphase multiplexed amino acid diagnostic array, which comprises (5) Boucher, J. L.; Charret, C.; Coudray-lucas, C.; Giboudeau, J.; Cynober, L. Clin. Chem. 1997, 43, 1421–1428. (6) Slocum, R. H., Cummings, J. G., Ed. Hommes FA. Amino acid analysis of physiological samples. Techniques in diagnostic human biochemical genetics: a laboratory manual;Wiley-Liss Inc: Somerset, NJ, 1991; pp 87-126. (7) Fuerst, P.; Pollack, T. A.; Graser, T. A.; Godel, H.; Stehle, P. J. Chromatogr., A 1990, 499, 557–569. (8) Sarwar, G.; Botting, H. G. J. Chromatogr., B 1993, 615, 1–22. (9) Fisher, G. H.; Arias, I.; Quesada, I.; D’Aniello, S.; Errico, F.; Di Fiore, M. M.; D’Aniello, A. Amino Acids 2001, 20, 163–173. (10) Jones, D. L.; Owen, A. G.; Farrar, J. F. Soil Biol. Biochem. 2002, 34, 1893– 1902. (11) Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clin. Chem. 2003, 49, 1797– 1817. (12) Sethuraman, R.; Lee, T. L.; Tachibana, S. Clin. Chem. 2004, 50, 665–669. (13) Burkholder, P. R. Science 1951, 114, 459–460. (14) Payne, J. W.; Bell, G.; Higgin, C. F. J. Appl. Microbiol. 1977, 42, 165–177. (15) Tuffnell, J. M.; Payne, J. W. J. Appl. Microbiol. 1985, 58, 333–341. (16) Chalova, V. I.; Kim, W. K.; Woodward, C. L.; Ricke, S. C. Appl. Microbiol. Biotechnol. 2007, 76, 91–99. 10.1021/ac100087r  2010 American Chemical Society Published on Web 04/20/2010

16 different amino acid E. coli auxotrophs yielding rapid, specific, and sensitive cell growth as a direct response to the concentration of corresponding amino acids. The extent of the specific cell growth stimulated by a specific amino acid is expected to be proportional to the concentration of the amino acid present in an unknown sample. To enhance the resulting cell-based signal, bioluminescence-based measurement of cell density was employed. As a result, an amino acid specific, concentrationdependent luminescence response is produced. The preliminary clinical validity of this approach was verified by reliably determining the concentrations of amino acids in newborn human blood specimens. EXPERIMENTAL SECTION Construction of Amino Acid Auxotrophs. Amino acids auxotrophs were constructed by transposon mutagenesis and NTG mutagenesis. Transposon Mutagenesis. One microliter of KAN-2 transposon (Epicenter, WI) was electroporated into Escherichia coli W (ATCC1105)13 using standard procedures.17 Transposon insertion mutants were selected on a Luria-Bertani (LB) plate containing Km (50 µg/mL). For selection of amino acid auxotroph among the Tn5-inserted mutants, the mutants were selected by replica plates, first on a M9 media plate containing Km and corresponding each amino acid and then on a M9 media plate containing only Km. After selection of candidate mutants for each amino acid auxotroph on the above plate selection, we confirmed the auxotrophic character of candidates in 3 mL of M9 liquid media with and without the corresponding amino acid, respectively. Using this Tn5 random transposition, Cys, Phe, His, Ile, Lys, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr auxotrophs were constructed. NTG Mutagenesis. Gly, Pro, and Ser auxotrophs were created by combination of Tn5 random transposition and NTG mutagenesis, because their candidates generated from KAN-2 mediated random transposition did not show enough auxotrophic specificity. For NTG mutagenesis of the candidate strains, each overnight culture of the strains was diluted 100 times, added into a fresh 5 mL of LB broth, and grown to exponential phase (OD600 ) 0.5). After washing twice with 0.1 M sodium citrate buffer (pH 6.0), NTG solution was added to a final concentration of 250 µg/ mL into 5 mL of the culture and then incubated for 30 min at 37 °C. The NTG-treated cells were harvested and washed four times with fresh M9 media. The pellets were resuspended with 1 mL of LB media and incubated for 1 h at 37 °C. The culture was washed twice with M9 media and screened on M9 media plates with and without the corresponding amino acids, respectively. Construction of pTAC-luc. To construct pTAC-luc, the T7lac promoter of the pETDuet-1 vector (Novagen, San Diego, CA) was replaced by a tac promoter, generating pTAC, and luc was amplified with the polymerase chain reaction (PCR) from pGL3Basic vector (Promega, WI) using a pair of primers, NcoI-luc-F (5′ CATGCCATGG AAGACGCCAAAAACATAAAGAAAGGC-3′) and EcoRI-luc-R (5′-CG GAATTC CTA GAA TTA CAC GGC GAT CTT TCC GC-3′). The amplified luc fragments, after digestion with NcoI and EcoRI, were cloned into pTAC that had been digested (17) Reznikoff, W. S.; Goryshin, I. Y.; Jendrisak, J. J. Methods Mol. Biol. 2004, 260, 83–96.

with the same enzymes, producing pTAC-luc. Plasmid was electroporated into each amino acid auxotroph using a Gene Pulser system (Bio-Rad, CA). Primers were synthesized from Bioneer (Daejeon, Korea). Construction of the Amino Acid Array and Assay of Amino Acids. After each amino acid auxotroph harboring pTAC-luc was cultivated in 2 mL of LB overnight, the cell cultures were centrifuged at 8000 rpm at 4 °C for 5 min. The individual cell pellets were washed twice with M9 minimal media, distributed into 2 × 106 cells per well, and mixed with 3% low melting agarose (Lonza, ME) as a 1:1 volume ratio. After mixing, 100 µL of the cell-agarose mixtures was deposited within the corresponding 20 wells of 96 well plates (Nunc, Roskilde, Denmark). After the mixtures were solidified at room temperature for 20 min, amino acid samples dissolved in 100 µL of M9 media containing amino acid cocktail or 5% of human blood plasma, 1 µL of 100 nM cyanocobalamin, and 1 µL of 100 mM IPTG were added to each of the wells. The amino acid cocktail consisted of 100 µM alanine, 50 µM arginine, 15 µM asparagine, 7.5 µM aspartate, 50 µM cysteine, 25 µM glutamine, 15 µM glutamate, 100 µM glycine, 25 µM histidine, 15 µM isoleucine, 25 µM leucine, 50 µM lysine, 7.5 µM methionine, 15 µM phenylalanine, 25 µM serine, 50 µM threonine, 15 µM tyrosine, 10 µM tryptophan, 50 µM valine, and 50 µM proline. Blood samples from healthy volunteers were taken at a local hospital and collected into tubes containing heparin. The blood was kept for 30 min at room temperature and then centrifuged at 6000 rpm at 4 °C for 30 min. The supernatant, serum, was collected and stored in aliquots at -70 °C until use. Luminescence was measured using a luminometer (Perkin-Elmer, MA), and scanning images were collected using a cooled charge coupled device camera (Fujifilm, Japan) with a constant focal plane, magnification, and integration time. Specificity Test of Amino Acid Array to Individual Amino Acid Solution. Sixteen individual auxotroph arrays were prepared to verify the target amino acid specificity of the constructed E.coli auxotrophs. Each array consisted of 20 of the same wells containing the same auxotroph and all 20 different amino acid solutions, containing 1 nM cyanocobalamin, 1 mM IPTG, and 10 µM of each amino acid in M9 media, were applied into each of the 20 wells of an individual array. After 4 h incubation at 37 °C, luminescence was measured. Effects of Incubation Time on the Linearity between Luminescence and Amino Acid Concentration. The tryptophan auxotrophs were deposited on 36 wells (6 × 6) using the same method as described above. Six kinds of amino acid cocktail solutions that consisted of a fixed concentration of 19 amino acids (100 µM alanine, 50 µM arginine, 15 µM asparagine, 7.5 µM aspartate, 50 µM cysteine, 25 µM glutamine, 15 µM glutamate, 100 µM glycine, 25 µM histidine, 15 µM isoleucine, 25 µM leucine, 50 µM lysine, 7.5 µM methionine, 15 µM phenylalanine, 25 µM serine, 50 µM threonine, 15 µM tyrosine, 50 µM valine, and 50 µM proline) and six different concentrations of tryptophan (0, 1, 2.5, 5, 7, and 10 µM) were prepared in M9 media containing 1 nM cyanocobalamin and 1 mM IPTG. During incubation for 6 h, 100 µL of each prepared media solution was added into each of the six wells at an interval of 1 h, and luminescence responses of all 36 wells were measured. Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

4073

Specific Linearity between Luminescence and Target Amino Acid Concentration. Four amino acid (isoleucine, lysine, arginine, and tyrosine) auxotrophs were deposited within 6 wells in a line, respectively. Four kinds of amino acid cocktails were prepared as described above, which contained various concentrations of isoleucine (0, 0.5, 1.5, 3, 7, and 10 µM), lysine (0, 6, 10, 14, 18, and 36 µM), arginine (0, 0.5, 2, 3, 4, and 6 µM), and tyrosine (0, 5, 10, 20, 30, and 50 µM), respectively. After four amino acid arrays were incubated with each solution above at 37 °C for 4 h, luminescence was measured. The specificity of all of the other 12 amino acid auxotrophs were also experimented as described above. Similarly, four amino acid (isoleucine, lysine, arginine, and tyrosine) auxotrophs were deposited within 6 wells in a line, respectively. Four kinds of blood plasma solutions were prepared. The intact blood plasma was diluted 1 in 20 (5%) in M9 media containing 1 nM cyanocobalamin and 1 mM IPTG, and six different concentrations of amino acids (isoleucine, lysine, tyrosine, and arginine, respectively) were also added. After incubation for 4 h, luminescence was measured. The specificity of all of the other 12 amino acid auxotrophs were also experimented as described above. Correlation between Amino Acid Array and the Conventional HPLC. The conventional HPLC method for amino acid determination was performed at DBVT (Daejeon, Korea) according to the manufacturer’s instructions and protocols. Isoleucine, lysine, arginine, and tyrosine solutions were prepared in M9 media and used for the analysis. The correlation between amino acid array and HPLC amino acid analyzer (Waters, MA) was measured using a isoleucine solution of 0-13 µM, lysine solution of 0-45 µM, arginine solution of 0-50 µM, and tyrosine solution of 0-16 µM. Determination of Amino Acid Concentrations from Clinical Dried Blood Spot Specimens. Dried blood samples of newborns were obtained from Labgenomics (Seoul, Korea). A paper disk, 5/16 in. in diameter, was used to prepare a standard curve. A 20 µL standard amino acid solution in cocktail was spotted on the filter paper and dried at room temperature. After drying, the paper was stored in a plastic envelope. The amino acid concentration of each spot equaled the added amino acid plus that presented in the original blood sample as determined previously.18 We punched the blood spot specimens (3 mm diameter) into wells of an amino acid array. The specimens included standard, control, and unknown real clinical samples. To elute amino acid adsorbed on the paper, samples were incubated with 3% trichloroacetic acid for 1 h at room temperature with moderate shaking. After this, 30 µL of eluted solution was mixed with 90 µL of reaction mix. The reaction mix was composed of 1 mL of 4× M9 minimal medium, 1 mL of sodium bicarbonate (1 M), 0.92 mL of sterilized water, 40 µL of IPTG (100 mM), and 40 µL of cyanocobalamin (100 nM). The mixed solution was inserted into the wells of the amino acid array to get the luminescence after incubation for 4 h. RESULTS AND DISCUSSION E. coli Auxotroph-Based Amino Acid Array. The key element of this study is a set of amino acid E. coli auxotrophs, (18) McCaman, M. W.; Robins, E. J. Lab. Clin. Med. 1962, 59, 885–890.

4074

Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

wherein growth is only possible in the presence of a corresponding amino acid, and is proportional to amino acid concentration. The amino acid array was designed to determine quantitatively the concentrations of 16 different amino acids (Figure 1). To develop the amino acid array, 16 genetically engineered amino acid auxotrophs of E. coli were constructed via a Tn5 random transposition system coupled with NTG mutagenesis. The targeted amino acids in this study included 10 essential amino acids (Phe, His, Ile, Lys, Leu, Met, Arg, Thr, Val, and Trp) and six aminoacidopathic indicators (Phe, Tyr, Met, Leu, Ile, Val) along with Gly, Pro, Ser, Glu, and Cys. The signal from cell growth was obtained using a firefly luciferase bioluminescent reporter gene supplemented to the E. coli auxotroph, which yields a bioluminescent response proportional to the concentration of the corresponding amino acid. The bioluminescence-generating plasmid pTAC-luc was constructed by inserting the luciferase gene (luc) derived from Photinus pyralis (firefly) into pET-pTAC containing an IPTG-inducible promoter and transformed to the auxotroph. The presence of a specific amino acid results in the proportional cell growth of the corresponding auxotroph after IPTG induction and consequently produces a bioluminescence signal without the requirement of an external substrate. The amino acid array was constructed by mixing each of the 16 E. coli auxotrophs with 1.5% (w/v) agarose and depositing the cells into respective wells of a 96-well plate. After 20 min at room temperature, the agarose solutions containing cells solidified, resulting in the amino acid array capable of assaying the aforementioned 16 amino acids. Evaluation of the Amino Acid Array: Specificity, Linearity, and Correlation with Conventional Methods. To verify target amino acid specificity of the constructed E. coli auxotrophs, we prepared 16 individual auxotroph arrays. Each array consisted of 20 identical wells containing a single auxotroph. All 20 different amino acid solutions were applied into each of the 20 wells of an individual array (Figure 2a). For all 16 auxotroph arrays after a 4 h incubation at 37 °C, luminescence was observed only in the well where the targeted amino acid was provided while the other 19 wells did not produce a significant signal. Thus, successful construction of the 16 E. coli auxotrophs was achieved, confirming the target specificity of the amino acid arrays (Figure 2b). Linearity between the luminescence signal and target amino acid concentration is essential for accurate quantitative analysis. Therefore, we proceeded to investigate response linearity during 1-6 h of incubation by employing a model tryptophan auxotroph. An assay time of > 4 h was necessary to achieve a linear dose response between Trp concentration and luminescence signal, with R2 ) 0.97 (Supporting Information, Figure S1). In addition to the Trp auxotroph, similar experiments were performed with the other 15 amino acid auxotrophs, all giving excellent linearity during 4 h of incubation (Figure 3a1 and Supporting Information, Figure S2). This short incubation time (4 h) is a great benefit of our system considering that the previously reported cell-based assay takes up to 40 h of incubation.14-16 The detection range is from 1 µM to several tens of µM, which is sensitive enough to reliably diagnose metabolic diseases of newborns related with amino acid concentrations in human blood. To enhance the sensitivity or widen the detection range, further efforts, including

Figure 1. Overall scheme to determine the concentrations of amino acids based on amino acid array. The amino acid array was constructed by mixing each of the 16 E.coli auxotrophs harboring the bioluminescence plasmid pTAC-luc with 1.5% agarose and depositing it in the respective well of a 96 well plate followed by solidifying it for 20 min at RT. For the multiplex analysis of amino acids, test sample (amino acid solution, human blood plasma, or extracted mixture from dried blood paper) was applied into each of the 16 wells and the luminescence signals were simply measured after 4 h of incubation at 37 °C. From the linear calibration curve between target amino acid concentration and bioluminescent response directly generated by the growth of the cells on the wells, amino acid concentrations of an unknown sample could be determined simultaneously.

Figure 2. Specificity of amino acid array for target amino acid. (a) Illustration of positions on the array applied by 20 different amino acid solutions. (b) Vivid target specificity of the 16 amino acid arrays. Twenty different amino acid solutions (10 µM) were applied into each of the 20 wells of an individual amino acid array.

expanded mutagenesis, would be required. Such efforts are underway in our group. The linearity of the amino acid array was also assessed on a clinical sample containing 5% human blood plasma. All 16 E. coli auxotrophs revealed high linearity (R2 > 0.96) (Figure 3a2 and Supporting Information, Figure S3), sufficient to consider use in a clinical setting.5,19 Additionally, the precision of the analysis for blood plasma was evaluated by calculating the coefficient of (19) Dietzen, D. J.; Weindel, A. L.; Carayannopoulos, M. O.; Landt, M.; Normansell, E. T.; Reimschisel, T. E.; Smith, C. H. Rapid Commun. Mass Spectrom. 2008, 22, 3481–3488.

variation (CV); [CV(%) ) SD/average × 100] of the luminescence signals obtained from four representative amino acids (Ile, Lys, Leu, and Met). As clearly indicated in the Supporting Information Table S1, all assay-assay precision of the amino acid arrays had CV < 8%, within an acceptable range when compared with those reported for currently used methods.5,19 Finally, we compared our array approach with a conventional HPLC method using the selected four amino acids (Ile, Lys, Arg, and Tyr) (Figure 3b). Various concentrations of the corresponding amino acids were applied, measured, and analyzed using both the amino acid array and HPLC. The observed correlation between the two methods was R2 > 0.99. This high concordance between the two methods ensured the validity of the amino acid array for reliable quantification of amino acid concentration. Clinical Use of the Amino Acid Array with Dried Blood Spot Specimens. Several amino acid concentrations in human blood are closely related to the state of inherited metabolic diseases of newborns.20 For example, higher than normal concentrations of Phe and Tyr are indications of potential phenylketonuria; high concentrations of Leu, Ile, and Val are associated with maple syrup urine disease (MSUD), and high Met levels in human blood are used to diagnose homocystinuria. Thus, significant incentive exists for the development of a rapid and efficient method to diagnose inborn errors of metabolism by determining specific amino acid concentrations from clinical blood samples, typically dried blood spot specimens. To that end, we evaluated the clinical applicability of our E. coli auxotroph-based amino acid array method to diagnose inborn errors of metabolism by determining the concentrations of amino acids eluted from the dried blood specimens provided by clinical hospitals. (20) Garg, U.; Dasouki, M. Clin. Biochem. 2006, 39, 315–332.

Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

4075

Figure 3. Linearity of amino acid auxotrophs to the concentrations of target amino acids and correlation with the conventional HPLC. Specific linearity of the auxotrophs for four selected amino acids (isoleucine, lysine, tyrosine, and arginine) to the concentrations of the corresponding amino acids. Luminescence (RLU) responses of the four auxotrophs to various concentrations of corresponding amino acids, respectively, were measured in an amino acid cocktail solution containing 19 other amino acids (a1) and a clinical sample containing 5% human blood plasma (a2). (b) Correlation between amino acid array (y) and conventional HPLC (x).

Following procedures described in Online Methods, after extracting amino acids from dried blood on specimen paper using 3% trichloroacetic acid (TCA) solution (Supporting Information, Figure S4), the concentrations of three selected Met, Phe, and Tyr amino acids were determined to diagnose newborn disease, and their detection precisions were evaluated (Table 1). For each amino acid, two independent blood specimens were tested in at least six parallel analytical sets. As a result, within-assay precision of amino acid array was below 7% and assay-assay precision 4076

Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

(CV%) run over 5 days was below 8% in all experimental data sets. Such precision was considered to be within an acceptable range.21,22 Blood specimens containing higher concentrations of Phe or Met than the cutoff values of newborn diseases, phenylketonuria and homocystinuria, were diagnosed using the amino acid array (21) Deng, C.; Deng, Y. J. Chromatogr., B 2003, 792, 261–268. (22) Nagy, K.; Takats, Z.; Pollreisz, F.; Szabo, T.; Vekey, K. Rapid Commun. Mass Spectrom. 2003, 17, 983–990.

Table 1. Detection Precision of Amino Acid Array for the Determination of Three Selected Amino Acids from Dried Blood Spot Specimens amino-acid assay

methionine sample 1

phenylalanine sample 2

sample 1

average (µM) SD CV (%)

17.8 0.9 5.4

17.9 0.8 4.6

Within-Assay Precision 20.3 1.3 6.8

average (µM) SD CV (%)

17.2 1.3 7.4

17.4 1.0 5.9

Between-Assay Precision 19.5 1.5 7.6

tyrosine

sample 2

sample 1

sample 2

18.9 0.8 4.1

33.2 1.2 3.7

30.5 1.1 3.8

18.1 1.3 7.4

31.8 2.3 7.1

29.2 2.1 7.3

and sensitivity. This method can also be extended to evaluate or monitor nutritional conditions, as the metabolic pathway of E. coli holds many metabolites.

Figure 4. Diagnosis of newborn disease with dried blood specimens by amino acid array. Diagnosis of phenylketonuria (a) and homocystinuria (b) by measuring luminescence from phenylalanine and methionine auxotrophs incubated with an eluted mixture from clinical blood paper specimens.

(Figure 4). As a result, there were vivid luminescence differences between the blood specimens from patient and normal samples. Thus, we conclude that the amino acid array method may serve as a promising analytical tool to diagnose inborn errors of metabolism related to specific amino acid concentrations in the clinic. Cell-based biosensors are commonly used for toxicity monitoring in environmental engineering, the food industry, and pharmaceutical screening.23 The advantages of these biosensors include convenient, rapid, and economical detection of potential toxicity, which otherwise would require specific instrumentation or time-consuming analysis. Cell-based biosensors have also been exploited to diagnose human diseases, such as phenylketonuria24 or galactosemia25 using auxotrophic bacteria through a well-known Guthrie test. However, these methods are often inaccurate and require significant analysis time. On the other hand, the amino acid array presented herein, which employs rapidly growing E. coli mutants coupled with a bioluminescence reporter, not only circumvents these traditional limitations but also quantifies physiological amino acid concentrations with higher clinical specificity (23) Pancrazio, J. J.; Whelan, J. P.; Borkholder, D. A.; Ma, W.; Stenger, D. A. Ann. Biomed. Eng. 1999, 27, 697–711. (24) Guthrie, R.; Susi, A. Pediatrics 1963, 32, 338–343. (25) Levy, H. L.; Hammersen, G. J. Pediatr. 1978, 92, 871–877.

CONCLUSIONS To our knowledge, this is the first report of a solid-phase amino acid array consisting of E. coli amino acid auxotrophs equipped with bioluminescent reporter enabling rapid (4 h) and convenient simultaneous quantification of multiple amino acids in a given sample. We further demonstrated high specificity and sensitivity of the amino acid array and also near-perfect linearity, which are sufficient for reliable quantification. In addition, excellent correlation between the amino acid array and currently used HPLC methods suggest that the array approach may be employed to clinically diagnose newborn metabolism errors due to abnormal specific amino acid concentrations. Such an approach may also be extended to other rapid cell-based sensing of simple metabolites that are relevant to other clinical disorders, including homocysteine, galactose, various vitamins, folic acid, and lipids. One of the challenges to accelerate practical applications of this cell-based biosensor would be to develop technical methods that enable convenient long-term preservation of the gel-based format without drying and, hence, loss of cell viability. ACKNOWLEDGMENT This work was supported by the grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2009-0080602), the Center for Ultramicrochemical Process Systems by KOSEF, and BK21. SUPPORTING INFORMATION AVAILABLE Figures for the linearity of amino acid auxotrophs to the concentrations of corresponding amino acids and multiple replicates of extracting amino acids from real blood spot specimens and a table for the detection precision of the amino acid array. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 12, 2010. Accepted March 31, 2010. AC100087R

Analytical Chemistry, Vol. 82, No. 10, May 15, 2010

4077