Development of colorimetric cholesterol detection kit using TPU nanofibre/cellulose acetate membrane

Abstract

In this study, the authors report a simple fabrication of thermoplastic polyurethane (TPU) nanofibres‐based kit for cholesterol detection. TPU is a polymer that is highly elastic, resistant to microorganisms, abrasion and compatible with blood; thus, making it a natural selection as an immobilisation matrix for cholesterol oxidase (ChOx) enzyme. The nanofibre was fabricated by electrospinning process and was characterised using scanning electron microscopy and Fourier transform‐infrared spectroscopy. ChOx was covalently immobilised on TPU nanofibre and cholesterol level/concentration was visually found using 4‐aminoantipyrine, a dye that reacts with H₂ O₂ produced from the oxidation of cholesterol by ChOx and changes colour from yellow to red. The efficacy of the nanofibre to act as a detecting substrate was compared with cellulose acetate (CA) membrane, a well‐documented enzyme immobilisation matrix. The optimisation of enzyme concentration and dye quantity were performed using standard ChOx spectrophotometric assay and the same was used in CA membrane and TPU nanofibre. The ChOx immobilised nanofibre showed good linear range from 2 to 10 mM with a lower detection limit of 2 mM and was highly stable compared to that of CA membrane. The enzyme immobilised nanofibre was further validated in serum samples.

1 Introduction

Cholesterol is a sterol biomolecule synthesised by cells to maintain structural integrity, fluidity of the cell membranes and is also a precursor for bile acids, vitamin D, and various steroid hormones [1]. The normal range of cholesterol is <5.1 mM and the border levels lie between 5.1 and 6.21 mM [2]. Although cholesterol is required for human body on a daily basis, the level beyond 6.21 mM is dangerous as it is associated with numerous diseases like atherosclerosis, coronary artery disease, cerebral thrombosis and hypertension [3]. Hence a continuous monitoring of cholesterol is needed for the clinical diagnosis of above mentioned diseases.

Numerous methods have been explored to determine the cholesterol concentration in blood. It includes spectrophotometry [4], high performance liquid chromatography [5], Colorimetry [6] and electrochemical methods [7]. Spectrophotometry is easy to perform but involves a large number of chemicals. Chromatographic methods are reliable, selective and accurate because the interference from other sterols is absent. However, its efficiency depends on the extraction procedures and the saponification steps. Electrochemical methods have a short response time, high sensitivity, low detection limit and good linear range but they are very complex and have reproducibility issues. Colorimetric methods are highly promising because they do not require any high precision equipment as in the other techniques. In principle non‐invasive techniques which employ colorimetry not only has the advantage of easy detection but also cost effective. The colorimetric sensors developed so far are based on nanomaterials that show surface plasmon resonance effect [8] or that mimic horse radish peroxidase (HRP) [9, 10]. The unearthing of the potential of nanomaterials to act as enzyme mimics started with Fe₃ O₄ nanoparticles, which opened up a new area of research for nanoparticles [11]. In a similar way, CO₃ O₄ nanoparticles showed an HRP enzyme like catalytic properties for the detection of glucose [12]. Various carbon based nanomaterials have been explored including graphene oxide [13], carbon nanotubes [14], graphene dots [15] and carbon dots [16] for their catalytic activity in the detection of H₂ O₂. However, they suffered from a lack of sensitivity, poor catalytic activity, low specificity compared to the enzyme‐based methods and complex to synthesise. [17]. Owing to the current detection drawbacks a simple, reliable and cost effective method needed to be developed.

The high surface area‐to‐volume ratio of the electrospun nanofibres makes it a promising material for sensor fabrication. These nanofibres have been explored to a great extent due to its ease of incorporating active agents and flexibility [18]. Thermoplastic polyurethane (TPU) is used in medicine for its good compatibility with blood [19]. It possesses a range of attractive properties like flexibility, resistant to microorganisms, abrasion, good hydrolytic stability and can be easily electrospun [20]; thus, making TPU a natural selection as an immobilisation matrix for cholesterol oxidase (ChOx) enzyme in the colorimetric detection of cholesterol. Cellulose acetate (CA) membrane, on the other hand, is used as an enzyme immobilisation matrix for glucose sensing [21]. It is hydrophilic in nature and the stability of alcohol oxidases in the CA membrane is also reported earlier by Murtinho et al. [22].

To the best of our knowledge, colorimetric detection kits based on membranes and nanofibres have not been reported to date.

The potential of this method to be used by simple observation lies in the direct relation between the concentration of cholesterol and the colour shade. Here the current work proposes the use of TPU nanofibre/cellulose acetate membrane as substrates for immobilisation and provides a colour wheel for the increasing concentration of cholesterol, which could be used as a band/strip to determine cholesterol from blood serum.

2 Experimental

2.1 Materials

Potassium dihydrogen phosphate (KH₂ PO₄) (Merck, ⩾98%), di‐potassium hydrogen phosphate (K₂ HPO₄) (Merck, ⩾98%), cholesterol (Sigma Aldrich), ChOx (Sigma Aldrich, 17 U/mg), HRP (Sigma Aldrich, 150 U/mg), 4‐aminiantipyrine (Sigma Aldrich, ⩾98%), phenol (LobaChemie, ⩾99.5%), EDC (1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride) (HiMedia, ⩾99%), N‐hydroxysuccinimide (NHS) (Fluka, ⩾97%), TPU (Covestro, Texin 945U grade), dimethylformamide (Merck, ⩾99%), CA membrane (Whatman^TM No 1, GE Healthcare UK Ltd, 0.45 µm, 110 mm) were purchased and used as received. All the solutions were prepared using Millipore water.

2.2 Preparation of TPU nanofibre

The TPU nanofibre was prepared by electrospinning process. In brief, the polymer solution was prepared by dissolving 1.5 g of TPU in 10 ml of N‐dimethylformamide followed by heating at 60°C in a hot plate with vigorous stirring. The prepared polymeric solution was electrospun (Indigenous Electrospinning Unit, PSGIAS, India) onto an aluminium plate. The nanofibres were electrospun at 20 kV in a working distance of 15 cm using syringe needle (21 gauge) at a flow rate of 0.2 ml/h. The collection time was kept as 2 h [23].

2.3 Characterisation of TPU nanofibres

The electrospun nanofibres were subjected to morphological characterisation using SEM (ZEISS EVO 18) with LaB6 as electron source and the images were captured at an accelerating voltage of 10 kV. The Fourier transform‐infrared (FTIR) spectroscopic analysis was carried out to determine the functional groups in the nanofibre using Schimadzu, IR Affinity where the spectrum was recorded using ATR mode fitted with ZnSe crystal.

2.4 ChOx assay

The protocol for ChOx assay was adopted from the previous literatures and was scaled down to microlitre reaction [24]. The parameters like concentration of enzyme, a concentration of 4‐aminoantipyrine (4‐AAP) and the time of incubation were optimised in the spectrophotometric method. The procedure, in brief,, involves the preparation of a 200 µl reaction mixture containing 0.1 M phosphate buffer saline, 5 mM cholesterol, 150 U/ml HRP, 1.76% AAP and 6% phenol. To the mixture 8 µl of ChOx enzyme was added. The solutions were mixed and incubated at 37°C for 5 min. Then the absorbance was recorded at 500 nm. The experiment was performed in triplicates.

2.5 Cholesterol assay

The optimised concentration of dye, enzyme and the time of incubation were used to perform the standard cholesterol assay where the concentration of cholesterol varied from 2 to 10 mM. The same assay was repeated with cholesterol spiked serum samples. All the experiments were done in triplicates.

2.6 Immobilisation of ChOx on CA membrane/TPU nanofibre

CA membrane/TPU nanofibre was cut into pieces with dimension 1 cm × 1 cm approximately. 15 µl of 0.2 M EDC and 0.05 M NHS in the ratio 3:4 was added to the CA membrane/TPU nanofibre and left for drying at room temperature. Then 8 µl of ChOx was added to the CA membrane, incubated at room temperature for 2 h and kept overnight at 4°C [25].

2.7 Cholesterol assay on CA membrane/TPU nanofibre

To the ChOx immobilised CA membrane, 25 µl of the reaction mixture containing an increasing concentration of cholesterol (2–10 mM) was added and the change in colour intensity was observed after 1 h of incubation. The same procedure was repeated with cholesterol spiked (2–10 mM) serum samples.

2.8 Reproducibility tests for immobilised TPU nanofibre

The ChOx immobilised CA membrane/TPU nanofibres were verified for their stability till 7 days with 10 mM cholesterol spiked serum samples.

3 Results and discussion

3.1 Nanofibre morphological characterisation

The TPU nanofibre produced by electrospinning was studied using scanning electron microscopy (SEM). Fig. 1 a clearly shows the formation of TPU nanofibres. The nanofibres were smooth and continuous with an average diameter of 298.7 nm and the fibre diameter range was between 149.39 and 413.16 nm. Fig. 1 b shows the EDS plot further confirms that the electrospun fibre is polyurethane. The FTIR spectra of TPU (Fig. 2) showed characteristic peaks at 1688 and 3330 cm⁻¹ which reflects the essential stretching vibration of the carbonyl and amino groups in TPU. The peak at 2930 cm⁻¹ is attributed to the asymmetric stretching of the CH groups. The obtained results were in agreement with other reported literature [26].

Fig. 1.

SEM image of

(a) Control TPU nanofibre and (b) EDS plot

Fig. 2.

FTIR pattern of control TPU nanofibres

3.2 ChOx assay

This assay is an indirect method to quantify cholesterol. The 3‐OH group of cholesterol is oxidised in the presence of ChOx enzyme to form cholest‐4‐en‐3‐one and H₂ O₂. The H₂ O₂ produced is quantitatively measured in a peroxidase catalysed colorimetric reaction where H₂ O₂ couples with 4‐AAP and phenol to produce a red colour 4‐quinoneimine dye as shown in Fig. 3. The dye intensity measured spectrophotometrically gives the cholesterol concentration [24].

Fig. 3.

Reaction mechanism for colorimetric detection of cholesterol

To investigate the influence of dye and enzyme in the development of colour, two variations of 4‐AAP (8, 12 µl) were tested with increasing concentrations of enzyme (0.2–1 U/ml).

An increasing absorbance profile was achieved for increasing enzyme concentration in both the dye variations. Comparing the spectrophotometric plots of 8 and 12 µl AAP, it is evident that increasing the concentration of 4‐AAP to 12 µl did not show much significant increase in the colour intensity and also 8 µl of 4‐AAP gave a linear plot for colour intensity after 1 h of incubation. Hence 8 µl of 4‐AAP was taken as an optimised concentration. From Figs. 4 and 5, it is also clear that 0.6 U/ml of enzyme produced substantial colour intensity relatively compared to other concentrations. Currently, available colorimetric cholesterol assay kits employ 0.2 U per assay to detect cholesterol [27, 28, 29]. In the proposed method, 8 µl of 0.6 U/ml solution containing 0.024 U of ChOx enzyme was used which is almost ten times less than the enzyme quantity that is used in commercial cholesterol assay kits. Fig. 6 gives a standard calibration plot for the spectrophotometric detection of cholesterol using the optimised protocol. A cholesterol assay with the optimised parameters was performed with cholesterol spiked serum samples to study the influence of serum interference in the assay. From Fig. 7, it is observed that with the increase in concentration, the absorbance increases. By increasing the time of incubation, the absorbance increases but the profile remains the same. As a result, depending on the observable colour change, 1 h of incubation time was fixed.

Fig. 4.

Spectrophotometric plot

(a) For 8 µl AAP with concentration of enzyme varying from 0.2 to 1 U/ml, (b) Corresponding plate image

Fig. 5.

Spectrophotometric plot

(a) For 12 µl AAP with concentration of enzyme varying from 0.2 to 1 U/ml, (b) Corresponding 96‐well plate image

Fig. 6.

Spectrophotometric plot

(a) For standard cholesterol assay, (b) Its corresponding plate image

Fig. 7.

Spectrophotometric plot

(a) For 2–10 mM cholesterol spiked serum samples taken at different incubation period, (b) Corresponding plate image

3.3 Immobilisation of ChOx on CA membrane/ TPU nanofibre

EDC is a crosslinker that reacts with the carboxyl group to form an O‐acylisourea intermediate. This intermediate when in contact with an amino group, it quickly forms an amide linkage and releases an isourea by‐product. The O‐acylisourea intermediate is unstable in aqueous solutions and gets hydrolysed easily if it fails to react with an amine. In order to prevent the hydrolysation, the intermediate is stabilised by the use of NHS, which forms an NHS ester that is stable and can react with an amine group. The NHS stabilised amine reactive intermediate forms an amide bond with the primary NH₂ group present in the ChOx enzyme. The NHS as such is not involved in the crosslinking reaction but acts as a stabiliser and also increases the enzyme coupling efficiency.

3.4 Cholesterol assay on CA membrane/TPU nanofibre

The cholesterol determination was done as shown in Fig. 3. When reaction mixture containing different concentrations of cholesterol was added to the ChOx immobilised CA membrane/TPU nanofibre, the reaction occurs (according to reaction 1 stated earlier) forming a red coloured quinoneimine dye product which is visually observed. Fig. 8 shows the change in intensity of colour with increasing cholesterol concentrations. Both the substrates showed increasing intensity with cholesterol concentration. TPU nanofibre, in particular, showed better intensity because the reaction is a surface phenomenon, whereas in CA membrane the active agents were absorbed into the membrane due to its hydrophilicity producing less colour intensity.

Fig. 8.

Standard cholesterol assay in

(a) CA membrane, (b) TPU nanofibre

Fig. 9 clearly depicts that the intensity of the colour is more in serum samples than a standard cholesterol due to the fact that serum itself contains 30% of cholesterol in free form. This serum cholesterol together with the added cholesterol produces a higher colour intensity as observed in spectrophotometric methods earlier. It is also apparent from Fig. 9 that the nanofibre is able to produce better results than CA membrane similar to the standard cholesterol assay. This further affirms that the nanofibre is a superior immobilisation matrix and can produce an improved colour change which can be easily visualised. The nanofibre was able to show a linear range from 2 to 10 mM with a detection limit of 2 mM which was comparable with other colorimetric biosensors as shown in Table 1.

Fig. 9.

Cholesterol assay in

(a) CA membrane, (b) TPU nanofibre for cholesterol spiked serum samples

Table 1.

Comparison of performance of various cholesterol biosensors

Material used	Method of detection	Linear range, M	Limit of detection ×10−3 M	Ref
ChOx/MWCNT–Au/chitosan/ IL	amperometry	0.5–5 × 10−3	0.35	[30]
MWCNT‐PANI	amperometry	1.29–12.93 × 10−3	—	[31]
NiO/MWCNT	amperometry	2.59–5.18 × 10−3	0.03	[32]
ChOx/peroxidase	colorimetry	0–7 × 10−3	—	[33]
ChOx/haemoglobin	amperometric	10–600 × 10−6	9.5 × 10−3	[34]
ChOx/molecularly imprinted polymer	impedance	5–30 × 10−6	0.42 × 10−3	[35]
ChOx/Amino‐undecanethiol SAM	SPR	1.295–12.95 × 10−3	—	[36]
ChOx/N‐(2‐aminoethyl)‐3‐aminopropyl‐trimethoxysilane SAM	spectrophotometry	1.29–2.93 × 10−3	—	[37]
ChOx/PtOEP loaded alginate‐silica microspheres	fluorimetry	1.25–10 × 10−3	1.25	[38]
ChOx/graphene/PVP/PANI	amperometry	0.05–10 × 10−3	1 × 10−3	[39]
ChOx/TPU nanofibre	colorimetry	2–10 × 10−3	2 × 10−3	this work

3.5 Study on interfering agent

To study the performance of the ChOx immobilised CA membrane and TPU nanofibre in the presence of interfering agents, cholesterol spiked serum samples were used for the analysis. The possible interfering agents in this detection method are biochemical components in blood like ascorbate, uric acid, bilirubin, and triglycerides as reported by many cholesterol kits [27, 40]. The actual serum sample containing the above mentioned interfering ions was used to compare with a standard cholesterol solution. Table 2 depicts the normal levels of the interfering agents in serum and the acceptable levels in the total cholesterol detection kit.

Table 2.

Levels of interfering agents in serum and kits (27)

Interfering agent	Normal levels of serum, mg/dl	Acceptable levels of kit, mg/dl
ascorbate	0.5–1.5	3
uric acid	3.4–7.2	20
bilirubin	0.1–1.2	8
intralipid	1000	1000

3.6 Reproducibility studies

The ChOx immobilised CA membrane and TPU nanofibres stored at 4°C were tested for their reproducibility with a 10 mM solution for 7 days. The CA membrane retained its activity only for 3 days, whereas the TPU nanofibre was able to produce repeatable colour up to 7 days. The enzyme activity started reducing with reduced intensity of colour on further storage up to 10 days. Beyond 10 days the activity was lost completely. From this reproducibility results obtained, TPU nanofibres portray better immobilisation efficiency than CA membrane. This is attributed to the increased surface area to volume ratio, which enabled the fibre to hold more enzyme, thereby increasing the activity of the ChOx immobilised TPU nanofibre towards cholesterol for a prolonged period. In case of CA membrane, the protein binding capacity is very low as reported by many kinds of literature [41, 42]. This filters the enzyme rather than anchoring it to its surface and hence the loss of activity.

4 Conclusion

In summary, the ChOx assay was optimised for 4‐AAP, ChOx concentrations and time of incubation using a spectrophotometric method of cholesterol detection. The ChOx enzyme was covalently immobilised on the CA membrane and electrospun TPU nanofibres using the EDC/NHS technique. The optimised ChOx assay was performed on both the substrates. We observed that the ChOx immobilised TPU nanofibre produced better colour intensity in detecting cholesterol than that of CA membrane. Furthermore, TPU nanofibre was able to retain the activity of ChOx better than CA membrane. This indicates the potential use of ChOx immobilised TPU nanofibre as a diagnostic kit for the detection of cholesterol in blood serum which is an easy technique compared to the previously reported methods.