A Simple and Fast Fluorimetric Method for Thiamine (Vitamin B1) Detection by Au<sup>3+</sup>-Mediated Formation of Thiochrome

Research Article

Austin J Biosens & Bioelectron. 2015;1(1): 1004.

A Simple and Fast Fluorimetric Method for Thiamine (Vitamin B1) Detection by Au3+-Mediated Formation of Thiochrome

Yu Y, Ching YJ and Tan YN

Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), Singapore

*Corresponding author: Tan YN, Institute of Materials Research and Engineering, Agency for Science, Technology and Research (A*STAR), 3 Research Link, Singapore 117602, Tel: 65 68747091; Email: [email protected]

Xueji Zhang, Chemistry Department, College of Arts and Sciences, University of South Florida, East Fowler Ave, Tampa, Florida 33620-4202, USA.

Received: September 30, 2014; Accepted: January 30, 2015; Published: February 02, 2015


Developing an effective method for thiamine detection is of great interest in clinical analysis, food processing, and pharmaceutical industries. A hitherto unreported, simple and efficient method for thiamine detection is demonstrated in this study by using Au3+ to convert the initially non-fluorescent thiamine into intensely blue-emitting thiochrome spontaneously in alkaline condition. The formation of thiochrome was characterized and confirmed by the photoluminescence and mass spectra. The linear response region for thiamine detection was observed to be 1.0x10-9-1.0x10-3 M with a correlation coefficient of 0.994. The limit of detection was calculated to be 2.0x 10-6 M. The unique homogenous-phase Au3+-assisted fluorescence detection method as developed in this study is not only highly specific to thiamine, but also simpler and faster than the conventional spectrofluorimetric methods without the need of extra separation process of thiochrome, the addition of highly toxic ions and exogenous enzymes to accelerate the formation of thiochrome.

Keywords: Vitamin B1; Fluorimetric detection; Thiochrome; Gold ions


RDI: Reference Daily Intake; LC-MS: Liquid Chromatography Mass Spectrometer; IT: Ion Trap; TOF: Time-Of-Flight; LOD: Limit of Detection; GSH: L-Glutathione, Tripeptideγ-Glu-Cys-Gly


Thiamine, also known as vitamin B1, is a water-soluble, S-containing vitamin consisting of an amino-pyrimidine and a thiazole ring linked by a methylene bridge [1]. Thiamine is an essential nutrient which plays a vital role in carbohydrate metabolism, maintenance of neural activity, and prevention of beriberi [2]. Human beings cannot synthesize thiamine and thus can only obtain it from their diet. The Reference Daily Intake (RDI) of thiamine for an adult human is 1.4 x 10-3 g/d. Medical doses of vitamin B1 pills or vitamin B1-enriched food or drinks are good means to obtain sufficient thiamine to maintain proper functions of the nervous and cardiovascular systems of the body. It is therefore of strong interest in the development of a simple (in terms of sensor construction and operation), fast, and reliable method for thiamine detection in clinical analysis, food processing, and pharmaceutical industries.

Several different analytical methods have been developed for thiamine detection [3-12]. In particular, a spectrofluorimetric method based on detection of the fluorescent substance thiochrome, which is the oxidizing product of thiamine in the presence of various oxidants, is the most frequently used one due to its simple, fast and sensitive manner [4]. The typical oxidants for athiochrome reaction include hexacynoferrate (III) [11,12], Hg2+ [5,10], Cu2+ [6],and hydrogen peroxide [13,14]. However, each of these reported oxidants has their own limitations for efficient thiamine detection. When hexacynoferrate (III) is used as the oxidant, another separation step is needed in order to extract the fluorescent thiochrome from the aqueous phase to organic phase, or it will be quenched in the presence of hexacynoferrate (III). Although Hg2+ is an efficient oxidant for the thiochrome reaction without quenching the fluorescence, it is highly toxic and not suitable for used in the biological systems. While Cu2+ is not as toxic as Hg2+, it may give false response in the presence of other biomolecules (such as cysteine) due to the formation of other fluorescent species (e.g., Cu nanoclusters [15]) under similar reaction conditions.When hydrogen peroxide is used as the oxidant, additional reagents such as enzymes are required to make the thiochrome formation more efficient, because meantime hydrogen peroxide as well as other strong oxidants such as KMnO4also tends to convert thiamine into non-fluorescent thiamine disulfide. Peroxidase exacted from horseradish [13], or Kohlrabi (Brassica oleraceagongylodes) [14] has been demonstrated for the detection of thiamine. However, the need of extra reagent will definitely increase the cost and add more complexity to the sensor system.

To address the above mentioned issues, herein we demonstrate a hitherto unreported method of using Au3+ as oxidant for thiamine detection. In this method, non-fluorescent thiamine was facilely converted to intensely fluorescent thiochrome in the presence of Au3+ under alkaline conditions, which was confirmed by the photoluminescence and mass spectra. The Au3+-mediated formation of thiochrome was further used to detect thiamine in aqueous solutions. A linear response range of 1.0 x 10-9- 1.0 x 10-3 M was achieved and the limit of detection for thiamine was as low as 2.0x10-6 M. Compared to previous studies, the current method eliminates the need of extra separation steps, involvement of highly toxic ions, and requirement of exogenous enzymes. In addition, the current method is highly specific for thiamine detection, even in the presence of a wide range of common biomolecules and other metal ions. Presented below are the details of this investigation.

Materials and Methods


Gold (III) chloride solution (HAuCl4, 30 wt.% in dilute HCl), thiamine chloride (vitamin B1, 99 wt. %), biotin (vitamin B7, 99 wt. %), L-cysteine (97 wt. %), L-glutathione (98 wt. %), D-(+)-glucose (99.5 wt. %), cellulose, copper (II) chloride (97 wt.%), cadmium chloride (99.99 wt.%), zinc chloride (97 wt. %), nickel(II) chloride (98 wt.%), lead (II) acetate trihydrate (99 wt.%), mercury (II) acetate (99.999 wt.%), cadmium nitrate tetra hydrate (99 wt.%), iron(III) nitrate nonahydrate(98 wt. %), aluminium chloride (99.0 wt. %), sodium chloride (99 wt. %), potassium chloride (99 wt. %), and lithium chloride (99 wt.%) were purchased from Sigma-Aldrich. All reagents were used as received and without further purification. Ultrapure water with a specific resistance of 18.2 MΩ was used throughout the experiment.

Fluorescence detection of thiamine

Aqueous solutions of HAuCl4 (2.0x10-2 M, 2.5 x 10-5L) and thiamine (2.0 x 10-2M, 2.5x10-5 L) were mixed in 9.2x 10-4Lof ultrapure water. 2 min later, 3.0x 10-5L of NaOH (1 M) was added to adjust the solution pH as 12. The final product was collected 10 min later. A Shimadzu 2450 spectrometer and a TECAN infinite M200 plate reader were used to record the UV-vis absorption and photoluminescence spectra, respectively. A Shimadzu Liquid Chromatography Mass Spectrometer (LC-MS) equipped with an Ion Trap (IT) as well as Time-Of-Flight (TOF) technology was used to record the mass spectra of thiamine and thiochrome.

Quantification of thiamine

2.5 x 10-5L of HAuCl4 (2.0x 10-2M) was mixed with 2.5 x 10-5L of thiamine solution with varied concentrations. The mixture was topped up with ultrapure water to 9.2x 10-4L. 2 min later, 3.0x10-5 L of NaOH (1 M) was added to this mixture to adjust the solution pH as 12. The emission spectra were recorded for the resultant samples after 10 min of reaction.

Specificity and interference measurements

To investigate whether Au3+ interact specifically with thiamine among various other biomolecules, aqueous solutions of biotin, L-cysteine, L-glutathione, D-(+)-glucose, and cellulose were used instead of thiamine to react with HAuCl4. To investigate the interference of other biomolecules, aqueous solutions of biotin, L-cysteine, L-glutathione, D-(+)-glucose, and cellulose were additionally introduced after the addition of thiamine of the same concentration. The rest reaction conditions and instrumentations were the same. To investigate the interference of other metal ions, aqueous solutions of 12 other metal ions (K+, Li+, Al3+, Zn2+, Ni2+, Cu2+, Pb2+, Ca2+, Hg2+, Na+, Cd2+, and Fe3+) was added in a 1:1 ratio of M-to-Au3+ to the mixture of HAuCl4 and thiamine.

Results and Discussion

Figure 1a shows the conversion of thiamine (1) to thiochrome (2) in the presence of Au3+ in alkaline conditions. Thiamine was initially non-fluorescent in aqueous solutions (Figure 1b, blue line, and inset item 1). Upon mixing with Au3+ in alkaline conditions (pH 12), a new species exhibiting intense blue emissionat445 nm was spontaneously formed (Figure 1b, purple line, and inset item 2). It should be mentioned that the presence of both Au3+ and NaOH was necessary for the formation of the blue-emitting species. No blue emission was observed in the absence of Au3+ (Figure 1b, red line) or NaOH (Figure 1b, green line). Photo excitation and mass spectra were measured to further characterize the resultant blue emitting species. It showed a distinct peak at 370 nm of the excitation spectrum. Both the excitation and emission profiles were similar to those [14] reported for thiochrome previously, suggesting the formation of thiochrome. Mass spectrum further confirmed the formation of thiochrome. A base peak at 263. 11 was observed on the mass spectrum, which could be assigned as thiochrome attached with a proton [C12H14N4OS + H]+.The resultant thiochrome showed very intense blue emission with quantum yield of 11% (calibrated with quinine sulfate). As shown in Figure 2, the formation of thiochrome was fast and could be completed in 10 minutes, which allowed the rapid detection of thiamine. Moreover, the as-formed thiochrome was found to be stable for several days (data not shown) in the current system and no separation steps were needed. Therefore, the conversion of thiamine to thiochrome by Au3+ could be exploited for simple and fast fluorimetric detection of thiamine.