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Article Cite This: ACS Omega 2018, 3, 6617−6623
Selective Screening of Biological Thiols by Means of an Unreported Magenta Interaction and Evaluation Using Smartphones Francisco J. Ortega-Higueruelo and Fernando Langa* Instituto de Nanociencia, Nanotecnología y Materiales Moleculares (INAMOL), Universidad de Castilla-La Mancha, Avda. Carlos III s/n, Toledo 45071, Spain
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S Supporting Information *
ABSTRACT: The determination of biological thiols is considered to be of great interest because of the participation of these molecules in physiological disorders as indicators of their existence and progress, such as Alzheimer’s disease, cancer, and cystic fibrosis, among others. Optical techniques for the quantification of these analytes are superior to other techniques in terms of selectivity, sensitivity, and cost. We describe here a simple protocol to determine biological thiols by means of a colorimetric analysis based on a magenta color interaction with 5,5′-dithiobis(2-nitrobenzoic acid). This methodology leverages the technology developed in smartphones, and it can be transferred to a simple analytical procedure through the use of absorbent strips and a cell phone. Excellent characteristics were observed, and this method opens up alternative avenues for the detection of biological thiols in a very simple and portable manner.
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INTRODUCTION Thiols that are present in living organisms include glutathione, both reduced and oxidized (GSH and GSSG, respectively), cysteine (Cys), and homocysteine (Hcy). These compounds participate in several physiological processes and play vital roles in many biological systems.1−4 These low-molecular weight thiols are involved in the pathogenesis of important disorders. For instance, Cys instils functions that include detoxification, synthesis of proteins, metal binding, and metabolism, and unusual levels of this essential amino acid could result in liver damage, skin lesions, slow growth, depigmentation, or edema.5,6 GSH is the most abundant thiol in biological systems, and it acts as an efficient defense against toxins and free radicals, thus protecting cellular components from reactive oxygen species.7,8 Abnormal levels of GSH could be due to a broad range of ailments because of the intense oxidative conditions for cells, with heart problems and cancer being two of the most common causes of demise.9 Given the importance of thiols from the biological, clinical, and environmental points of view, there is growing interest in the development of novel analytical methodologies to quantify thiols. High-performance liquid chromatography,10,11 gas chromatography,12,13 mass spectrometry,14−16 capillary electrophoresis,17,18 electrochemical methods,19,20 and nuclear magnetic resonance spectroscopy21 are diverse techniques used with the goal of detecting biological thiols. Nevertheless, these methods suffer from certain drawbacks associated with the intrinsic practical complexity, for example, high cost of the equipment and maintenance, processing of results, potentially complex chemical derivatizations required on the sample, and analysis time, which can be excessively long in cases where time is a critical factor. These issues mean that some of the © 2018 American Chemical Society
aforementioned techniques are either inconvenient for routine clinical self-analysis or analyses performed in field research because the analysis system must be portable and low-cost and enable rapid analysis times. A promising alternative to the approaches outlined above is the development of chemical entities that are suitable for use as optical sensors to detect and measure targeted biological thiols by colorimetric and fluorometric spectroscopy. These analytical techniques have a lower associated cost, shorter measurement times, easier operation, and the instrument can be transported to areas in which the analyst needs to carry out the determination rapidly. In recent years, a wide variety of fluorogenic substrates, considering both the generation of light with a specific wavelength after target detection and the quenching of energy for the same interaction with the analyte, have been presented and described in the scientific bibliography on the detection of biological thiols.22−27 Given the widely reported utility of these fluorescent probes to quantify thiols, usually for tissue imaging in medicine, there has been tendency to neglect the search for colorimetric, nonfluorometric methodologies. However, the advantages of the aforementioned alternatives are inherently associated with the simplicity of use, low cost, portability of the equipment, and excellent sensitivity. Several decades ago, Ellman described a simple and valid method for the measurement of the sulfhydryl content in tissue by using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB).28 Tietze subsequently developed Ellman’s work and identified an enzymatic method for the Received: April 21, 2018 Accepted: June 7, 2018 Published: June 20, 2018 6617
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ACS Omega quantitative determination of total and oxidized glutathione.29 This approach is currently considered the method of reference to determine GSH and GSSG, with kits commercially available from numerous chemical and pharmaceutical companies. Despite this situation, this particular method for quantification has some disadvantages related to the use of an enzymatic cycle (glutathione reductase is required for the reduction of GSSG to GSH mediated by nicotinamide adenine dinucleotide phosphate, NADPH). This shows a gradual loss of activity for the enzyme and consequently can have an adverse effect of the accuracy of the method. Furthermore, the cost of this method is relatively high, and it is therefore desirable to search for alternative methods to overcome the drawbacks outlined above. We report here a simple nonenzymatic optical methodology to detect and quantify thiols for common analysis in biological samples. The method is based on a previously undescribed colorimetric response by DTNB beyond the commonly used measurement at 412 nm due to the release of the TNB− anion in Tietze’s approach. In this new strategy, a chemical derivatization to mask GSH and remove it from the total titration of thiols to quantify oxidized species is not required. In this new method, the reduced thiols react in a nonenzymatic manner with DTNB in a specific solvent, dimethyl sulfoxide (DMSO), to yield a previously unreported DTNB-mediated magenta coloration in the solution. This color is suitable to be measured by any smartphone application capable of identifying color space (in particular the CMYK code).
enzymes, there is a lack of reproducibility associated with the clear variability of their catalytic activities depending on the ambient preservation of these proteins. To design our experiments for the alternative method, we questioned why an efficient direct determination is not available for thiols in the absence of enzyme and utilizing the minimum number of chemical components in the cuvette (analyte + sensor + solvent). It was decided to evaluate the route in the cycle at the bottom of Scheme 1. The choice of solvent was made to achieve complete solubility of both GSH and DTNB. As a consequence, deionized water, chloroform, ethanol, and DMSO were tested because of their appropriate polarities for oligopeptides. However, only the last solvent was capable of dissolving GSH and DTNB completely and separately. Unexpectedly, on adding to a vial the combination of DMSO as solvent, DTNB as the eventual oxidant, and GSH as the analyte to be evaluated, a different behavior was observed to that represented in Scheme 1 (i.e., a yellow color visible to the naked eye). In our case, the behavior after combination was characterized by an attractive red color (Figure 1a). None of the other solvents tested produced a similar effect, including the previously demonstrated cleavage of DTNB to yield yellow TNB− in the presence of GSH. The selectivity of the detection was evaluated by testing other biological thiols and amino acids, such as GSSG (pure, without free thiol), Cys, Hcy, glycine (Gly), alanine (Ala), arginine (Arg), and proline (Pro). A colorimetric response was not observed for GSSG, but the addition of a biological reducing agent, that is, nicotinamide adenine dinucleotide (NADH), which is significantly cheaper than NADPH, led to the appearance of an intense color. The reducing conditions clearly promoted cleavage of the disulfur bond and the subsequent interaction with DTNB/DMSO in a similar way to that observed with the GSH sample (Figure 1a,b). On investigating other thiols, it was found that Cys also gave a marked response. Both GSH and Cys gave rise to a maximum absorbance at 499 nm (Figure 1c). GSSG gave a peak at 484 nm after reduction. The remaining amino acids did not give rise to optical responses between 450 and 500 nm. GSSG itself did not exhibit signals at these wavelengths, as one would expect given the presence of the disulfur functional group. The different analytes shown in Figure 1a were evaluated at several concentrations to develop the corresponding features of the sensing methodology. The colorimetric response of the system DMSO/DTNB was studied in the presence of the analytes. Measurements were made at room temperature, and the wavelength was established at 499 nm. Moreover, the spectrophotometer worked under the “timecourse” operating mode and therefore did not register any isolated absorbance. This was applied as a result of the increase of the optical signal with time (Figure S1 in the Supporting Information). Thus, the values represented in Figure 1d correspond to the maximum difference in the absorbance between the blank and the top value registered for each concentration. It can be seen from Figures S1 to S9 (Supporting Information) that the curves fit parabolic, linear, or decreasing behaviors, depending on the nature of the analyte. For thiols that were capable of giving a response, namely, GSH, GSSG, and Cys, the corresponding detection limits (LODs) were calculated to measure the smallest concentration that could be determined with a specified precision, working as a function of both sensitivity and signal stability.30 The LOD values were as
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RESULTS AND DISCUSSION The enzymatic cycle that is commonly employed to assess the amount of total thiols, both reduced and oxidized, is shown in Scheme 1. This procedure is not entirely rigorous because Scheme 1. Conventional Spectrophotometric DTNB Protocol to Determine Total Biological Thiols Mediated by a Redox Enzymatic Cycle29
GSH is considered to be the only thiol present in biological tissues, which is not strictly the case. However, other thiols such as Cys and Hcy are only present at low levels, and it is widely accepted that total thiols (including Cys and Hcy) are currently determined by the enzymatic cycle shown in Scheme 1. Nevertheless, when developing a method to detect the presence of chemical compounds with the participation of 6618
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Figure 1. (a,b) Distribution of colorimetric signals for the biological thiols analyzed, considering GSH, GSSG, and GSSG after reduction with NADH, Cys, and Hcy, and other amino acids with potential interferences, such as Gly, Ala, Arg, and Pro. All samples were first prepared with distilled water at a concentration of 40 mM, and they were then diluted to 10 mM in DMSO. Front view of samples (a) and top view (b). (c) Absorption spectra obtained from the analysis of the abovementioned samples diluted to a final concentration of 0.4 mM in DMSO. (d) Regression for optical responses for different concentrations of thiols and nonthiols. Error bars (mainly negligible) represent standard deviation (n = 3).
follows: LODGSH = 15.6 × 10−9 M; LODGSSG = 41.6 × 10−9 M; LODCys = 36.4 × 10−9 M, improving sensitivities associated with other remarkable works based on fluorescence.31 This strategy avoids the inconvenience of derivatization of GSH where the GSSG/GSH ratio must be calculated by colorimetric techniques (e.g., Ellman’s reagent in Scheme 1). In essence, the global amount of total oxidized and reduced thiols is determined and GSH is conveniently derivatized and thus removed from the probe, with only the GSSG present in the sample being measured by enzymatic methods. In contrast, on applying our methodology, one can initially determine the GSH concentration at 499 nm and then apply reducing conditions and determine the growth of the absorbance at 484 nm, as shown in Figure 1c. This new strategy was evaluated by preparing a range of mixtures of GSH and GSSG with the aim of checking the direct titration at 499 and 484 nm, respectively. Probes that contained a constant concentration for GSH and variable GSSG concentrations were analyzed optically, and the data are plotted in Figure 2a. First, GSH was determined at 499 nm in the presence of DMSO and DTNB. The sample was then treated with NADH to reduce GSSG, and the measurements were then made at 484 nm. As one would expect, the GSH values remained practically unchanged on account of the fixed concentration at 5.5 μM. The reductive effect of NADH on GSSG led to an increase in the absorbance (the graph is provided in the Supporting Information, Figure S11). NADH did not show an absorbance at 499 or 484 nm (Supporting Information, Figure S12) and therefore did not interfere in the thiol determination. Tests incorporating other antioxidants and reducing agents of the common presence in biological samples, such as N-acetylcysteine, ascorbic acid, and glucose, were conducted, and no optical effects on GSSG determination were noted by means of the application of our methodology. This easy sequential determination of GSH and GSSG is a promising alternative method that does not require chemical
derivatization of GSH to remove it from the system, a drawback that is inherent in contemporary procedures.32 It seems clear that an interaction occurs between the components in the mixture. Magnetic resonance measurements were performed to identify the functional groups that participate in the generation of the magenta color (Figure 2b). DTNB signals showed a pronounced variation as the concentration of GSH increased, with an attenuation and shift of sensor signals observed until they vanished completely as the signals due to the thiol increased in intensity in proportion to its concentration. Next, we applied this methodology to determine the GSH concentration present in a real human serum commercially purchased. We used a standard addition procedure considering different GSH concentrations (Supporting Information, Figure S13). The average GSH concentration in the serum was determined considering absorbance differences (Figure 2c) and found to be 1.035 ± 0.025 μM (n = 3). The average percent recovery of our method (defined as ([GSHestimated]/ [GSHtheoretical]) × 100) was determined in 95%. The consequent comparison to the reference methodology33 was achieved to validate our determination, depicting similar concentrations. The portability of any method for chemical analysis is a key aspect in terms of development and potential applicability, such that it can be implemented in field studies away from the laboratory or in any situation without the need for cumbersome equipment usually used in laboratories. Finally, the cost associated with the analysis is an important aspect, and this must be minimized by controlling the investment in laboratory equipment. An interesting option in this respect is to harness the adaptability of smartphones, which are now ubiquitous in our society. The recent development of smartphones and their impressive technological applications open a staggering variety of options in numerous areas. Userfriendly smartphone analytical methods are continuing to emerge, and some examples have already been described in the 6619
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components of any tonality in the color range. Only the magenta percentage was considered owing to its predominance over the others. The procedure was utilized to determine the presence of GSH in a number of samples. Tests on different concentrations of GSH (0−1000 μM) were carried out in glass vials along with aluminium strips that had an area of 0.5 cm × 0.5 cm with permeable silica (Figure 3a). The strips were immersed in GSH solutions in an appropriate position for 2 min until complete permeation of the silica zone with GSH solution had been achieved (Figure 3b). The strips were immediately taken out of the vials and treated with several drops of DTNB solution (0.1 mM) on the active surface (Figure 3c). The color changed immediately (Figure 3d), and after 1 min, the active zones for quantification generated a gradient in magenta tonality depending on the concentration. Data registration was performed by focusing on up to 10 different points within the active zone for each strip to guarantee a suitable statistical data translation (Figure 3e and Table S1 in the Supporting Information). Finally, the resultant graph demonstrates the surprising capacity of this simple methodology to register a large accumulation of data and achieve excellent capacities in conjunction with great accuracy, simplicity, speed, and low cost (Figure 3f). The exponential behavior includes a linear region with an upper limit of around 80 μM GSH. The LOD was determined to be 11.4 × 10−6 M.
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CONCLUSIONS In summary, we have developed an innovative way to determine thiols such as GSH, Cys, and the oxidized species GSSG. The approach is based on an unprecedented colorimetric interaction, which has not previously been reported in the literature, between thiols and DTNB in the presence of DMSO, and this change is suitable for evaluation at a specific wavelength. Excellent characteristics were identified in terms of selectivity and sensitivity in the presence of other biological molecules or in real samples, and this method is useful for the direct quantification of GSH/GSSG ratios without the need for chemical modifications. This strategy has been successfully extrapolated to allow the portable analysis of thiols using the camera included in a smartphone and a suitable app for color detection. Good results were obtained, and this opens new doors for the use of smartphone technology for the titration of a wider variety of analytes.
Figure 2. (a) Responses of several mixtures of GSH and GSSG in the presence of NADH in a reductant role. Measurements were made at two different wavelengths depending on the thiols. Each system contains DTNB 1.4 mM in DMSO. (b) 1H NMR shifts depend on the variable GSH concentration in the system. (c) GSH concentration in human sera estimated by the standard addition procedure. Error bars for (a,c) represent standard deviation (n = 3).
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EXPERIMENTAL SECTION Materials. Commercially available materials were used without further purification. DTNB, DMSO, L-cysteine (Cys), DL-homocysteine (Hcy), glycine (Gly), L-alanine (Ala), DLarginine (Arg), L-proline (Pro), ascorbic acid, and human sera were purchased from Sigma-Aldrich. NADH was purchased from Roche. L-Glutathione reduced (GSH) and L-glutathione oxidized (GSSG) were obtained from Acros Organics. Silica strips were prepared manually from silica gel 60 with 0.20 mm of thickness placed on aluminum sheets (20 cm × 20 cm) for thin-layer chromatography. These silica sheets were purchased from Macherey-Nagel. Deionized water, chloroform, and ethanol were supplied by Scharlab. Methodology. All experiments were conducted at room temperature (between 20 and 25 °C). Optical experiments were performed on a Shimadzu 1603 UV−vis spectropho-
bibliography for pH measurement34 or anion detection,35 thus confirming a contemporary development associated with more advanced algorithms for smartphones. In terms of colorimetry, a broad range of software applications, commonly called apps, are available for users for various platforms, such as Android, iOS, BlackBerry OS, or Windows Phone. In the work described here, the recently distributed free app Hex Color Detector version 0.0.2, developed by Senseify for Android 5.0 Lollipop, was employed as a surprisingly accurate color identifier by means of alphanumerical codes. More specifically, the CMYK encryption proved to be the most appropriate for our purposes, where cyan, magenta, yellow, and black (key color) are the 6620
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Figure 3. Determination of GSH samples using a smartphone. (a) GSH solutions from 0 to 1000 μM in DMSO and strips with a square area of permeable silica. (b) Strips were immersed until liquid saturation was achieved. (c) Strips treated by pipette deposition of DTNB in DMSO on the active silica surface. (d) Magenta coloration appears after a short period of time. (e) Color treatment of the image, focusing the detector on the sensor surface. (f) Colorimetric response derived from the app analysis, representing the magenta parameter in the CMYK code dependent on the GSH concentration (in μM). The exponential fit resulted in the equation y = 41.459(1 − e−0.005x) with R2 = 0.995. Meanwhile, the linear range was adjusted to the equation y = −2.126 + 0.221x with R2 = 0.993. Error bars represent standard deviation (n = 10).
tometer and quartz cuvettes with 1 cm of width and 3 mL of volume. Two registration modes were utilized for spectrophotometry. First, a scanning mode served to identify the wavelengths of interest for subsequent experiments (Figure 1c). Second, experiments were carried out using the timecourse mode of the spectrophotometer to control the evolution of absorbance in relation to time and resulting absorbance variations (Figures 1d and S1−S9). The app for color detection was Hex Color Detector, version 0.0.2, from Senseify, available for Android 5.0 Lollipop and installed on an LG G2 D620 smartphone. The 1H NMR spectrum was recorded on a Bruker 400 MHz spectrometer at 20 °C. All 1H NMR spectra are reported in parts per million (ppm) downfield of TMS and were measured relative to the signals for CHCl3 (7.26 ppm). Statistical and graphics treatments were carried out using SigmaPlot 12 software by Systat (USA). Identification of Optical Activity and Interrogation of Different Samples (Thiols and Nonthiols). Diverse chemical analytes were interrogated in the spectrophotometer to investigate the maximum absorbance and sufficient selectivity in our procedure. Therefore, probes with GSH, GSSG, Cys, Arg, Gly, Ala, Hcy, and Pro were initially prepared using distilled water. The concentration in this stage for the stock solution corresponded to 40 mM. The next step involved
diluting these solutions to 10 mM in different solvents: distilled water, ethanol, chloroform, or DMSO. With the exception of DMSO as solvent, none of the other options allowed for the complete solution of thiols and amino acids, thus making DMSO the solvent of choice. On applying these conditions, solutions showed evident coloration (Figure 1a,b), and optical profiles for samples were registered from 300 to 700 nm, with peak maxima at 484 nm for GSSG reduced by NADH, while GSH and Cys presented intense peaks at 499 nm (Figure 1c). Arg gave a moderate peak at 403 nm, but this did not interfere with the measurements. Visualization of Optical Responses for Different Analytes and Determination of the LOD. Several aliquots of analytes (GSH, GSSG, Cys, Arg, Gly, Ala, Hcy, and Pro) were prepared at the following concentrations (μM) in cuvettes: 0.0, 0.1, 0.4, 1.0, 1.9, 3.0, 3.5, 5.0, and 5.5. In addition, the cuvette included DTNB 1.4 mM as a sensor reagent and DMSO as the solvent until the cuvette was full. Optical measurements were taken using the timecourse method in the spectrophotometer, given that the absorbance presented a parabolic profile that gave a maximum and then diminished. The graphics are included in this Supporting Information in the following order: Figure S1 for GSH, Figure S2 for GSSG, Figure S3 for GSSG treated with NADH, Figure 6621
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value in the absence of sera human was registered under the timecourse function of the spectrophotometer (Figure S13). Determination of Thiols by Means of a Smartphone Color App. GSH solutions (0.0, 10.0, 20.0, 40.0, 60.0, 80.0, 150.0, 250.0, 400.0, 700.0, and 1000.0 μM) in DMSO were prepared in vials along with handmade aluminum strips with an absorbent silica zone at the edge (Figure 3a). Each strip was immersed in the corresponding vial until complete permeation (Figure 3b). Immediately after, 50 μL DTNB 0.1 mM in DMSO was deposited in succession on each wet area (Figure 3c) and coloration appeared (Figure 3d). A photo was taken of each strip, and this was opened with the smartphone app (Figure 3e). In this work, ten different points were selected to identify the magenta index in the CMYK color code, but more points could be examined if required. To conclude, the magenta index was found to be dependent on concentration (Figure 3f) with five assays required for this determination.
S4 for Cys, Figure S5 for Hcy, Figure S6 for Ala, Figure S7 for Gly, Figure S8 for Pro, and Figure S9 for Arg. Differences in absorbance between the maximum value and the blank value were taken into consideration to generate Figure 1d after 3 assays for each concentration. Statistical fits gave the following equations from a linear regression GSH → y = −1.070 × 10−3 + 0.066x ;
R2 = 0.998
GSSG + reductant → y = −9.625 × 10−3 + 0.095x ; R2 = 0.998 Cys → y = − 2.472 × 10−3 + 0.016x ;
R2 = 0.965
Arg → y = − 2.881 × 10−4 + 2.376 × 10−3x ; R2 = 0.913
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Gly → y = 1.752 × 10−3 + 1.118 × 10−3x ;
ASSOCIATED CONTENT
S Supporting Information *
R2 = 0.951
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00783. Evolution of absorbance at a determined wavelength for every considered analytes according to a timecourse adjustment to quantify; 1H NMR spectrum for DTNB; sequential determination of GSH and GSSG at different wavelengths; lack of optical interferences for NADH in the area of interest; graphical representation of GSH determination in human sera by means of the standard addition procedure; and a table containing CMYK codes for determination by a smartphone (PDF)
Ala → y = 3.548 × 10−3 − 3.640 × 10−4x ; R2 = 0.252 Hcy → y = 1.076 × 10−3 + 1.147 × 10−4x ; R2 = 0.081 Pro → y = 1.044 × 10−3 + 4.761 × 10−4x ; R2 = 0.905
The LOD was calculated as following
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Limit of detection (LOD) = n(SD0 · n )
where n is the number of values or repetitions obtained for each concentration, and SD0 corresponds to the standard deviation of the lower concentration capable of being measured through a significant variation of the signal. Sequential Determination of GSSG/GSH Mixtures without Chemical Derivatization. Some aliquots containing GSH and different concentrations of GSSG were prepared. For GSH, a concentration fixed at 5.5 μM was established for all samples. However, the GSSG was included according to a gradual growth, namely, 0.0, 0.5, 1.0, 2.0, 3.0, and 4.0 μM. These reagents were examined by visible spectrophotometry in cuvettes, also as mixtures with DTNB (1.4 mM) and DMSO as solvent. The timecourse method in the detector was set up at 499 nm. The signal increases because of the existence of GSH until stabilization or a decrease (about 10 min). At this point, 2 mg of NADH powder was added to the cuvette, and the wavelength was changed to 484 nm. The signal then continued to increase before stabilizing again. A range of behaviors for the considered different aliquots are represented in Figure S11, and the evolution caused by consecutive increases in GSSG concentration is shown in Figure 2a. Determination of GSH in Commercial Sera Human. Powder sera human (1 mg) was solubilized in 0.5 mL of deionized water and 9.5 mL of DMSO and then homogenized. Separately, stock solutions of DTNB and GSH in DMSO were prepared. Finally, for each cuvette, 1.4 mM of DTNB and different concentrations of GSH (0.0, 0.1, 0.7, 1.9, and 3.5 μM) in cuvette were prepared in sera human solution as solvent. The difference between maximum absorbance and the
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (F.L.). ORCID
Fernando Langa: 0000-0002-7694-7722 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful for financial support from the Spanish Ministerio de Economiá y Competitividad (CTQ201679189-R). F.J.O.H. thanks the University of Castilla-La Mancha for a postdoctoral grant (77332274H) included in the Plan Propio UCLM Postdoctoral ProgramAcceso al Sistema Españ ol de Ciencia, Tecnologiá e Innovación (SECTI).
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DOI: 10.1021/acsomega.8b00783 ACS Omega 2018, 3, 6617−6623
