Development of a one-step analysis method for several amino acids using a microfluidic paper-based analytical device.

A one-step analysis method was developed for four types of amino acids using a microfluidic paper-based analytical device fabricated from chromatography filtration paper and laminate films. Aminoacyl-tRNA synthetase was used to detect each amino acid. The obtained laminated paper-based analytical device (LPAD) contained four enzymatic reaction areas. Colorimetric detection was performed based on the molybdenum blue reaction. A model method for the simple, easy, and simultaneous detection of several amino acid concentrations was suggested, in contrast to the conventional methods such as HPLC or LC-MS. The method provided a selective quantification at the ranges of 3.6-100 µM for tryptophan, 10.1-100 µM for glycine, 5.9-100 µM for histidine and 5.6-100 µM for lysine with a detection limit of 1.1 µM, 3.3 µM, 1.9 µM and 1.8 µM, respectively. LPAD fabrication was considerably simple, and the subsequent detection process was easy and required a short period of time (within 15 min).

Microfluidic Paper-Based Analytical Device for Histidine Determination.

A laminated paper-based analytical device (LPAD) for histidine detection was fabricated from a chromatography filtration paper and laminate films. Histidine recognition was effected by histidyl-tRNA synthetase (HisRS), and its detection was signaled colorimetrically based on the molybdenum blue reaction. The analytical conditions and detectable concentration range of histidine were examined. The method provided selective quantification from 1 to 100 µM histidine. LPAD fabrication is considerably simple, involving only the craft-cutting of the chromatography filtration paper and laminate film, and is cost-effective.

Effects of a remote mutation from the contact paratope on the structure of CDR-H3 in the anti-HIV neutralizing antibody PG16.

PG16 is a broadly neutralizing antibody to the human immunodeficiency virus (HIV). A crystal structure of PG16 revealed that the unusually long 28-residue complementarity determining region (CDR) H3 forms a unique subdomain, referred to as a "hammerhead", that directly contacts the antigen. The hammerhead apparently governs the function of PG16 while a previous experimental assay showed that the mutation of TyrH100Q to Ala, which does not directly contact the antigen, decreased the neutralization ability of PG16. However, the molecular mechanism by which a remote mutation from the hammerhead or contact paratope affects the neutralization potency has remained unclear. Here, we performed molecular dynamics simulations of the wild-type and variants (TyrH100Q to Ala, and TyrH100Q to Phe) of PG16, to clarify the effects of these mutations on the dynamics of CDR-H3. Our simulations revealed that the structural rigidity of the CDR-H3 in PG16 is attributable to the hydrogen bond interaction between TyrH100Q and ProH99, as well as the steric support by TyrH100Q. The loss of both interactions increases the intrinsic fluctuations of the CDR-H3 in PG16, leading to a conformational transition of CDR-H3 toward an inactive state.

Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli.

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5'-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p1, p4-di(adenosine) 5'-tetraphosphate, and adenosine-5'-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 µM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively). Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5'-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap4A, P1, P4-di(adenosine) 5'-tetraphosphate; AMP: adenosine-5'-monophosphate; PAR: pyrophosphate amplification rate.

Flow Analysis of Amino Acids by Using a Newly Developed Aminoacyl-tRNA Synthetase-Immobilized, Small Reactor Column-Based Assay.

Abnormal concentrations of amino acids in blood and urine can be indicative of several diseases, including cancer and diabetes. Therefore, analyses that examine amino acid concentrations are useful for the diagnosis of such diseases. In this study, we developed an enzyme-immobilized, small reactor column for flow analysis of amino acid concentrations. For the recognition of asparagine and lysine, asparaginyl-tRNA synthetase and lysyl-tRNA synthase were immobilized onto microparticles, respectively, and coupled with coloration reagents for spectrophotometric detection. This assay has some advantages in the analytical field, such as the ability to detect small amounts of analyte, allowing for the use of a small reaction volume, and ensuring a rapid and efficient reaction rate. This approach provided selective quantitation of up to 480 µM of asparagine and lysine in 200 mM Tris-HCl buffer (pH 8.0).

Chemiluminescence Detection of Serine, Proline, Glycine, Asparagine, Leucine, and Histidine by Using Corresponding Aminoacyl-tRNA Synthetases as Recognition Elements.

Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20 µM for serine, proline, glycine, and leucine; 1 to 60 µM for asparagine; and 1 to 150 µM for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics.

Microfluidic analysis of serine levels using seryl-tRNA synthetase coupled with spectrophotometric detection.

The measurement of amino acid content is useful for the diagnosis of several types of diseases, including cancer and diabetes. In this study, a microfluidic method for the analysis of serine using enzymatic reactions coupled with spectrophotometric detection was developed. The assay system has some advantages in the analytical field, such as the ability to detect small amounts of analyte and reaction solution and a rapid and efficient reaction. For the specific detection of serine, seryl-tRNA synthetase was coupled with the generation of hydrogen peroxide, which was then detected by the Trinder reagent spectrophotometric method. Seryl- and other aminoacyl-tRNA synthetases are involved in the biosynthesis of peptides and proteins in the human body and should allow precise recognition of the corresponding amino acids. This approach provided selective quantitation of up to 250 µM serine in 100 mM Tris-HCl buffer (pH 8.0) in a semiautomatic system.

Spectrophotometric detection of histidine and lysine using combined enzymatic reactions.

An amino acid-sensing system with absorption spectrophotometric detection was developed. To ensure specific recognition of each amino acid, aminoacyl-tRNA synthetases were employed and the concentration of NADH produced by way of several enzymatic reactions was measured. Using this detection system, from 1.5 to 55 µM of histidine and from 15 to 95 µM of lysine could be measured selectively in HEPES-KOH buffer (pH 8.0).

A luminol chemiluminescence method for sensing histidine and lysine using enzyme reactions.

The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl-tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid-aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0-30 µM histidine and 1.0-60 µM lysine.

Amino acid sensing using aminoacyl-tRNA synthetase.

The detection of amino acids using aminoacyl-tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50 microM tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable.

Selectivity and recovery performance of phosphate-selective molecularly imprinted polymer.

A phosphate-selective molecularly imprinted polymer was prepared using 1-allyl-2-thiourea as a functional monomer, and the binding ability and selectivity of the polymer were evaluated. The imprinted polymer showed high binding ability to and selectivity for phosphate in aqueous media. The recoverability of phosphate from the imprinted polymer was also examined, and nearly 70% of highly concentrated phosphate could be recovered.

Molecular recognition by indoleacetic acid-imprinted polymers--effects of 2-hydroxyethyl methacrylate content.

Molecularly imprinted polymers for indole-acetic acid were prepared by co-polymerizing N,N-dimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), and ethylene glycol dimethacrylate. The dependence of the affinity and selectivity of the imprinted polymers on HEMA content was evaluated chromatographically. The affinity was improved by increasing the HEMA content; the selectivity of the imprinted polymer was best when the HEMA content was approximately 30%, irrespective of monomer content.