Optical isopropanol biosensor using NADH-dependent secondary alcohol dehydrogenase (S-ADH)
Graphical abstract
Introduction
Isopropanol (IPA), also known as 2-propanol or isopropyl alcohol, is a colorless liquid with a bitter taste [1] and is the simplest secondary alcohol. IPA is an important solvent and commonly used in industrial activity such as a washing agent in the process of semiconductor manufacturing [2] and it is often found in the environment like in food additives, a component of furniture cleaner or as a solvent of some cosmetics [3]. Also, IPA is widely used in hospitals as the antiseptic alcohol gel for and disinfectant.
From a medical point of view, IPA rarely exists in the human body or only in very low concentration unless accidental ingestion or exposure to gaseous IPA [4], [5]. Recently, several studies reported that IPA could be measured in plasma or urine from ketoacidosis patients, who had not been exposed to or ingested IPA [6], [7]. For example, Petersen et al. pointed out that in the 75% of diabetic ketoacidosis (DKA) and 52% of alcoholic ketoacidosis (AKA) postmortems, IPA could be measured in the serum with a concentration of 15.1±13.0 mg/dL and 18.6±12.5 mg/dL [8]. Jessica B. Dwyer reported a chronic alcoholic patient case that IPA could be detected in the serum with a concentration of 6 mg/dl [9]. The researchers postulated that the serum IPA not only came from the ingestion but also may come from the metabolism of acetone by the enzymatic reaction of alcohol dehydrogenase (ADH) in some situations [8], [9], [10]. Usually, IPA would be metabolized to acetone in the liver by ADH or cytochrome P-4502E1 [11], [12], [13], however, in some situations, ADH will also convert acetone to IPA reversely due to the change of serum pH, excessive acetone or the elevated ratio of reduced nicotinamide adenine dinucleotide (NADH) to oxidase nicotinamide adenine dinucleotide (NAD+) [9], [10]. It seems there is potential that blood IPA can be a biomarker of DKA or to be the monitoring indicator for a diabetic because hyperglycemia is one of the most important factors causing DKA. Not only in humans, some studies also found that blood and rumen IPA concentration would increase in the ketotic dairy cows, which is an obsession for animal husbandry [14], [15]. Therefore, measuring IPA in the biological sample is necessary and valuable.
The conventional blood or urine IPA measurement methods include gas chromatography-flame ionization detector (GC-FID) [16], [17], [18], [19] or headspace gas chromatography with flame ionization detection (HS-GC–FID) [7], [8], [20]. These GC-FID based detection methods are highly sensitive but inconvenient to use, they need complicated pretreatment of samples and need high costs for a large amount of detection. An enzyme-based biochemical sensor provides an alternative candidate method for an accurate, simplified and low cost of IPA measuring. In our previous studies, we have already developed an optical biosensor using ultraviolet light-emitting diode (UV-LED), optical fiber probe, and photomultiplier tube (PMT) for ethanol determination [21]. The measuring principle of this ethanol sensor is based on the enzymatic reaction ADH that can catalyze ethanol to become acetaldehyde and meanwhile reduces co-enzyme NAD+, which acts as the electron acceptor, to be NADH. NADH comes with the unique optical property that when it absorbs 340 nm UV light, it will release visible fluorescence while NAD+ does not have same property [22]. Thus, the ethanol concentration is determined according to the fluorescence intensity emitted from the NADH [21], [23], [24], [25]. This kind of NADH-dependent optical biosensor is convenient to use, low cost and with quick response time. Besides, it is not only can be used for the ethanol determination but also for other compounds according to corresponding enzymes. For example, NADH-dependent secondary-alcohol dehydrogenase (S-ADH) comes with following catalyzing reaction equation:
S-ADH can catalyze isopropanol to be acetone and utilize NAD+ as the electron acceptor in alkaline condition. Therefore, an IPA biosensor also can be constructed according to this kind of technology and conceptions.
In this research, we constructed and evaluated a highly sensitive, which was based on the NADH-dependent S-ADH for the determination of IPA in the liquid phase. By the enzymatic reaction of S-ADH, relevant concentrations of NADH would be produced depending on the concentration of IPA. The produced NADH would be excited using a UV-LED system and the fluorescence would be calculated by the PMT. These two components were connected together using a two-way branch optical fiber and equipped with a fiber probe that was fixed with the enzyme-immobilized membrane. Construction and characterization of the IPA biosensor using S-ADH immobilized membrane and UV-LED excitation system would be described first. And then, the measurements of the IPA in nail related cosmetics products were also performed.
Section snippets
Reagents and chemicals
β-NADH were bought from Oriental Yeast, Co., Ltd., Japan and dissolved in deionized (DI) distilled water collected from Milli-Q purification system (Millipore, Co., USA) for several concentrations. NAD+ (β-NAD+: Oriental Yeast Co., Ltd., Japan) and NADH-dependent secondary alcohol dehydrogenase (S-ADH, EC. 1.1.1.x, 1 unit mg−1, E001) from yeast (Daicel Chiral Technologies, Co., Japan) were prepared in suitable buffer respectively and storage in the refrigerator before use. Hydrophilic
Characterization of the IPA biosensor
Results of NADH measurement are presented in supplementary Fig. S1. Fluorescence intensity increased immediately and reached to steady state in a short time when the standard NADH was dropped into the dark cuvette. This phenomenon revealed that the NADH measurement system had a fast response. 90% response time was within 2 min at 1 mol L−1. The calibration range for NADH detection was from 10 nmol L−1 to 10 mmol L−1 (R=0.997), and more details are described in the Supplementary information.
For
Conclusions
In this work, a UV-LED based fiber-optic NADH fluorescence measurement combined with S-ADH immobilized membrane for determination of IPA was developed, and demonstrated. The optimization conditions were characterized including buffer pH, temperature, and concentration of NAD+. This IPA biosensor performed calibration range from 500 nmol L−1 to 1 mmol L−1 with high selectivity. Compared to traditional detection methods, rapid response of the biosensor can be applied for large amount measurement in
Acknowledgments
This work was partly supported by Tokyo Medical and Dental University Scholarship (Sony Corporation supported), Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 26280053, by Japan Science and Technology Agency (JST) and by Ministry of Education, Culture, Sports, Science and Technology (MEXT).
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