Interfacing a Personal Glucose Meter with Cell-Free Protein Synthesis

Jan 22, 2019 - We developed a method to analyze amino acids using a personal glucose meter (PGM). In this method, the principles of protein biosynthes...
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Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

Interfacing a Personal Glucose Meter with Cell-Free Protein Synthesis for Rapid Analysis of Amino Acids Yeon-Jae Jang,† Kyung-Ho Lee,† Tae Hyeon Yoo,‡ and Dong-Myung Kim*,† †

Department of Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Korea ‡ Department of Molecular Science and Technology, Ajou University, 206 Worldcup-ro, Yeongtong-gu, Suwon 16499, Korea

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ABSTRACT: We developed a method to analyze amino acids using a personal glucose meter (PGM). In this method, the principles of protein biosynthesis were interfaced with the sensing mechanism of a PGM to enable simple and ubiquitous measurement of amino acids. A reaction mixture for cell-free protein synthesis was designed to synthesize a bacterial invertase in response to exogenous addition of a specific amino acid. The invertase synthesized upon addition of an assay sample containing the amino acid of interest was used to convert sucrose into glucose, which was detected using a PGM. The titers of the amino acid in assay samples were precisely represented by the readouts of a PGM. In addition to the convenience provided by use of a PGM, the accuracy and reproducibility of this method were comparable to those of standard high-performance liquid chromatography based methods.

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complemented with the missing amino acid. When the reaction mixture for this complementary cell-free protein synthesis (CCFPS) was programmed with the gene of bacterial invertase and mixed with an assay sample containing the amino acid, the amount of cell-free synthesized invertase was proportional to the level of the amino acid in the assay sample. Therefore, on the basis of the subsequent generation of glucose by cell-free synthesized invertase, we could quantify the amino acid using a PGM. This scheme enabled rapid determination of the amino acid titer and provided accurate and reproducible results that were comparable to those obtained by conventional high-performance liquid chromatography (HPLC) methods. In addition to its value for analysis of an important class of biomolecules, this method demonstrates how a fundamental biological mechanism can be used to convert levels of analytes into measurable signals compatible with a contemporary electrochemical sensing device.

mino acids are involved in many biological functions, including regulation of gene expression, cell signaling, and immune responses.1−3 The maintenance of optimal balances among the levels of individual amino acids is thus crucial for homeostasis, and abnormal levels of specific amino acids are often associated with various diseases. For example, abnormally high concentrations of certain amino acids are used as clinical indicators of metabolic diseases such as phenylketonuria,4 maple syrup urine disease,5 and hyperlysinemia.6 Moreover, recent studies revealed that changes in the amino acid profile in blood are related to various types of cancers.7,8 However, methods to analyze amino acids in biological samples have not markedly evolved, and conventional chromatographic techniques are still commonly used. The present chromatographic methods based on physical separation of derivatized amino acids are time consuming and require complicated instruments, which intrinsically limits the clinical analysis of assay samples. Thus, development of an alternative method that enables sensitive, rapid, and ubiquitous measurement of amino acids will substantially facilitate the diagnosis and management of amino acid related diseases. A generic method that enables routine measurement of amino acids would also be a valuable analytical tool in various industrial sectors, including the food, animal feed, drug, and nutritional supplement sectors.9,10 In this study, on the basis of the basic principle that amino acids are the building blocks of proteins, we designed a strategy to interface the molecular process of protein biosynthesis with the sensing mechanism of a personal glucose meter (PGM) to assay amino acids. In this scheme, a reaction mixture for cellfree protein synthesis (CFPS) was prepared without amino acids so that it can produce protein only when it is © XXXX American Chemical Society

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EXPERIMENTAL SECTION Preparation of an Amino Acid Free Cell Extract. As a source of translational machinery, an S12 extract was prepared from the Escherichia coli (E. coli) BL21Star(DE3) strain as described previously.11 Residual amino acids in the S12 extract were removed by ultrafiltration using a Vivaspin centrifugal concentrator (Sartorius Stedim Biotech GmbH, Göttingen, Germany) to prevent their interference with the measurement Received: November 29, 2018 Accepted: January 9, 2019

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DOI: 10.1021/acs.analchem.8b05526 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

proteins. Consequently, this method can be flexibly integrated with various signal-reading devices depending on the biological activity of synthesized proteins, in contrast with the conventional HPLC assay, which relies on column-based separation of individual amino acids. This study integrated this principle of CCFPS with the sensing mechanism of a PGM. A PGM is one of the very few detection devices that has been globally distributed at affordable prices, and consequently this portable apparatus is an ideal platform for ubiquitous and routine measurement of analytes. Most of the presently available PGMs detect electrons generated during oxidation of glucose into gluconolactone by glucose oxidase. To link the presence of amino acids in assay samples with measurement of glucose by a PGM, the reaction mixture for CCFPS was programmed with DNA encoding an invertase that catalyzes hydrolysis of sucrose into glucose and fructose. Neither sucrose nor fructose is recognized by glucose oxidase in the strip of a PGM; therefore, the enzymatic activity of a functional invertase can be determined by measuring the amount of glucose converted from sucrose. Using invertase as a reporter protein in the CCFPS reaction, the PGM readout was expected to represent the amino acid titer in the assay sample. The overall scheme of this approach is outlined in Figure 1. In a standard reaction mixture for CFPS, which contained all 20 amino acids, the concentration and solubility of invertase were approximately 640 μg/mL and 56%, respectively (Figure 2A). Performance of the CFPS reaction using the diafiltered

of amino acids in assay samples.12 After addition of 18 mL of wash buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesium acetate, 80 mM potassium acetate, and 1 mM dithiothreitol) to 2 mL of S12 extract, the concentrator was centrifuged at 2000g to return the diluted extract to its original volume (2 mL). After this step was repeated three times, aliquots of S12 extract were flash-frozen in liquid nitrogen and stored at −80 °C. CFPS Reactions. A standard reaction mixture for CFPS consisted of the following components in 30 μL: 57 mM HEPES-KOH, pH 8.2; 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 80 mM ammonium acetate; 12 mM magnesium acetate; 80 mM potassium acetate; 34 μg/mL 1,5-formyl-5,6,7,8-tetrahydrofolic acid; 2 mM each of 20 amino acids; 2% polyethylene glycol 8000; 3.2 U/mL creatine kinase; 67 mM creatine phosphate; 24% (v/v) diafiltered S12 extract; 6.7 μg/mL template DNA. The open reading frame of invertase (EC 3.2.1.26) was PCR-amplified from genomic DNA of the E. coli BL21 strain and cloned into the pK7 vector13 to create the pK7Inv plasmid, which was used as the template for CFPS reactions. For CCFPS reactions, 26 μL of the reaction mixture for CFPS was prepared without a target amino acid. The CCFPS reaction was performed by incubating the reaction mixture after mixing it with 4 μL of an assay sample that may contain the target amino acid. All the CFPS and CCFPS reactions were conducted for 1 h in a water bath set at 30 °C Use of Cell-Free Synthesized Invertase To Determine Amino Acid Levels. After completion of a CCFPS reaction, 15 μL of the reaction mixture was transferred to a microtube containing 15 μL of 100 mM sucrose solution in 50 mM Trisacetate buffer (pH 6.0). After incubation at 37 °C for 10 min, the enzymatic hydrolysis reaction was terminated by heating the reaction mixture at 90 °C for 10 min. After a brief centrifugation at 3000g, 5 μL of the supernatant was withdrawn and spotted onto a strip to measure the glucose titer using a PGM (Accu-Check Inform II, Roche Diagnostics, Mannheim, Germany). The PGM readouts were compared against a standard curve to determine the amino acid concentrations in the assay samples. For the assay of amino acids in fetal bovine serum (FBS), to inactivate the serum enzymes that interfere with protein synthesis, FBS was heated at 90 °C for 10 min before a CCFPS assay.12 Heat-aggregated proteins were removed by ultrafiltration using a centrifugal concentrator, and 4 μL of the filtrate was added to the reaction mixture for a CCFPS assay. The results of the CCFPS-PGM assay were verified using a Hitachi L-8900 amino acid analyzer (Hitachi High-Technologies, Tokyo, Japan) following the manufacturer’s protocols. SPSS Version 22.0 software (SPSS Inc., Chicago, IL) was used for statistical analyses of data. *p > 0.4 was considered statistically insignificant.

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RESULTS AND DISCUSSION Cell-Free Synthesis of a Functional Invertase. The CCFPS assay works by adding exogenous amino acids to a CFPS reaction mixture lacking the amino acid of interest.12 For example, a CCFPS reaction mixture lacking leucine fails to generate functional, full-length proteins. Only truncated proteins are produced because ribosomes stall at leucine codons. However, the reaction mixture can synthesize functional proteins when it is mixed with an assay sample that contains leucine. The levels of leucine in assay samples can be determined by measuring the activities of synthesized

Figure 1. Schematic illustration of the complementary cell-free translational assay for quantifying amino acids. B

DOI: 10.1021/acs.analchem.8b05526 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Cell-free synthesis of functional invertase. (A) A reaction mixture for cell-free protein synthesis was prepared without the template DNA (pK7Inv) and amino acids. The reaction mixture was incubated after adding the template DNA and/or 20 amino acids to measure the total (blank bars) and soluble (filled bars) amounts of cell-free synthesized invertase. (B) Varying amounts of cell-free synthesized invertase were added to 50 mM sucrose solution, and the mixture was incubated at 37 °C for 10 min. A 5 μL portion of the sample was withdrawn, and the amount of glucose generated from the hydrolysis of sucrose was measured using a PGM. Error bars represent the standard deviations of three independent experiments.

Figure 3. Standard curves prepared for concentrations of six amino acids vs PGM readout.

Use of CCFPS for PGM-Based Measurement of Amino Acids. On the basis of the above results demonstrating successful conversion of sucrose into glucose by cell-free synthesized invertase, we explored the use of invertase in the CCFPS assay to measure the levels of amino acids. Six amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, and tyrosine) were selected as primary target amino acids for analysis because they are related to various metabolic disorders (Table S1). Using standard amino acid solutions of various concentrations, we prepared linear standard curves of amino acid concentration against the PGM reading for the six selected amino acids (Figure 3). The glucose concentration was measured over a linear range of amino acid concentrations (up to 20 μM) for all six amino acids tested, and the limit of detection of the amino acids ranged from 0.1 to 1.5 μM. Because most of biological fluid contains glucose, before

S12 extract demonstrated that synthesis of invertase was strictly dependent on the presence of amino acids in the reaction mixture. For example, protein expression from the pK7Inv plasmid using the diafiltered S12 extract was negligible without exogenous addition of amino acids and was as low as that in the negative control reaction lacking template DNA (Figure 2A). This suggests that the basal levels of invertase activity and amino acids in the diafiltered extract were not high enough to interfere with the proposed assay scheme. Furthermore, we confirmed that cell-free synthesized invertase was highly functional. After completion of the CFPS reaction, varying amounts of cell-free synthesized invertase were added to sucrose solution, and 5 μL of the mixture was used to measure glucose titer after 10 min incubation. The PGM readouts showed a linear increase in proportion to the amount of invertase up to 200 μg/mL (Figure 2B). C

DOI: 10.1021/acs.analchem.8b05526 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry testing the CCFPS assay for the amino acid concentrations of biological samples, we examined if the presence of glucose in the reaction mixture for protein synthesis can interfere with the subsequent measurement of glucose generated by invertase. When a cell-free protein synthesis reaction was conducted in the presence of varying concentrations of glucose, it was found that the glucose was rapidly degraded during the cell-free synthesis reaction. When a cell-free protein synthesis reaction was conducted in the presence of glucose as high as 10 mM (150 mg/dL), no significant PGM readouts were obtained after 10 min of incubation. It should be noted that the cell-free protein synthesis system employed in this study is based on a crude extract of E. coli. Therefore, most of the cellular enzymes, including those involved in glucose metabolism, are also present in the reaction mixture for cell-free protein synthesis. Due to its metabolic consumption of glucose in the reaction mixture of cell-free protein synthesis, therefore, it was concluded that the glucose in assay samples would not affect the PGM measurement of glucose generated by cell-free synthesized invertase. We then tested the developed method to quantify amino acids in biological samples. FBS was used for this purpose as a surrogate for clinical human samples. When varying volumes of heat-treated FBS were added to incomplete cell-free synthesis mixtures (complete reaction mixtures lacking one of the six target amino acids) for the CCFPS assay, the PGM readouts increased in proportion to the volume of FBS used for cell-free synthesis reactions up to 4 μL (Figure 4A). The amino acid levels estimated by comparing the measured glucose concentrations with standard curves were almost identical with those determined by the standard HPLC assay, demonstrating the validity of this method (Figure 4B). Although the proposed method requires individual preparation of a reaction mixture for each of the target amino acids, it markedly reduces the assay time and instrumental complexity of amino acid analysis, which can be particularly useful in resource-poor settings.

Figure 4. Measurement of amino acids in FBS using PGM. (A) Varying volumes of heat-treated FBS were added to the reaction mixtures of CCFPS assay of six amino acids. When the amounts of glucose generated by the cell-free synthesized invertase were measured, PGM readouts demonstrated linear correlation with the volume of FBS used in the CCFPS assay up to 4 μL (filled triangles, Ile; open triangles, Leu; filled squares, Lys; open squares, Tyr; filled circles, Phe; open circles, Met). Error bars represent the standard deviations of three independent experiments. (B) Amino acid concentrations of FBS measured using the PGM (blank bars) were compared with the values obtained with the standard HPLC method (filled bars). *p > 0.4. Error bars represent the standard deviations of three independent experiments.

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CONCLUSION In this study, we developed a method to link the CCFPS technique with measurement of glucose by a PGM, which enabled rapid, convenient, and ubiquitous analysis of amino acids. This method consists of two steps: CCFPS of invertase and measurement of glucose generated from sucrose by the synthesized invertase. During the CCFPS reaction, functional invertase was produced in response to addition of exogenous target amino acids present in assay samples. Invertase subsequently produced glucose from sucrose in proportion to the titers of amino acids in the assay samples. Measurement of invertase-generated glucose by a PGM showed linearity over wide ranges of concentrations (up to 20 μM). The limit of detection of the amino acids ranged from 0.1 to 1 μM. Although a few studies have investigated the use of a PGM to analyze biomolecules other than glucose,14,15 these methods used antibody-conjugated invertase to produce glucose in an ELISA format.16 To the best of our knowledge, this is the first report describing the use of a PGM for homogeneous analysis of biomolecules. While six amino acids closely related to common metabolic diseases were examined in this study, in principle, this method can be extended to other amino acids. Some of the amino acids might be interconverted by the enzymes present in the cell extract (i.e., Asp and Asn, Glu and Gln, and Asp and Glu), which can interfere with the proposed

translation-based assay. The problem of enzymatic conversion of amino acids, however, should be readily solved by employing the PURE cell-free synthesis system that harnesses purified translational machinery.12,17

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications Web site. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b05526. Diseases associated with target amino acids studied in this work and degradation of glucose during cell-free protein synthesis reactions (PDF) D

DOI: 10.1021/acs.analchem.8b05526 Anal. Chem. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Author

*E-mail for D.-M.K.: [email protected]. ORCID

Tae Hyeon Yoo: 0000-0003-1448-3165 Dong-Myung Kim: 0000-0001-7875-0694 Author Contributions

T.H.Y. and D.-M.K. designed the experiments; Y.-J.J. and K.H.L. performed the experiments, and D.-M.K. supervised the work. Y.-J.J., T.H.Y., and D.-M.K. wrote the manuscript. All authors have given approval of the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (grant numbers 2015M3D3A1A01064878 and 2016M1A5A1027465).

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REFERENCES

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DOI: 10.1021/acs.analchem.8b05526 Anal. Chem. XXXX, XXX, XXX−XXX