Review—Enzymatic Strips for Detection of Serum Total Cholesterol with Point-of-Care Testing (POCT) Devices: Current Status and Future Prospect

With the quick development of sensing technologies for cholesterol detection, various detecting methods have been explored including surface plasmon resonance,⁵² fluorescence,⁵³ colorimetry,⁵⁴ and electrochemistry detection,⁵⁵ etc. Among these methods, colorimetry and electrochemical detection methods have the merits of low cost, high effectiveness and well-acceptance, which are attracting and desirable for POCT applications. Thus, these two methods still prevail in numerous researches and commercialized products for POCT in the past decades.

As the science of color quantification, colorimetry is essential for color reproduction technology. In the early nineteenth century, the study was begun with the work of Young, Helmholtz and Maxwell, who recognized the principles of additive and subtractive colour mixing, and proposed the trichromatic nature of human colour vision.⁵⁶ In 1931, the science became formalized, and further developed with the recommendation of the Commission International de l'Eclairage (CIE), which the system about color specification based on the three tristimulus values X, Y and Z (XYZ). The current CIE reference represents more practical information on colorimetric measurement.⁵⁶ Colorimetry method is the very traditional and classical method for enzymatic assays. The aim of this method is to numerical describe the colors quickly and accurately. It is particularly attractive for point-of-use applications because the measured results can be simply read out in a single sheet of paper by unaided eyes, or through the absorbance of the color produced. Perception of the color is governed by three factors: the nature of the illumination, the optical properties of the object itself and the response of the human eye. Typically, colorimetry quantitates these aspects to unambiguously define color using three primary colors (red, green and blue, RGB).^56,57

Cholesterol in the blood comes in two forms, free cholesterol (about 30%) and cholesteric esters (about 70%). The typical principle of enzymatic cholesterol colorimetric detection is shown as reaction Eqs. 1–3. At first, esterified cholesterol is hydrolyzed to free cholesterol by cholesterol ester hydrolase/cholesterol esterase (CEH). Subsequently the free cholesterol is oxidized to cholest-4-en-3-one and hydrogen peroxide (H₂O₂) by cholesterol oxidase (COD/ ChOx). H₂O₂ is produced in this reaction as a side product and can be easily detected using high sensitive colorimetric probes. The producing H₂O₂ results in increasing intensity of color produced with increasing concentration of analyte via oxidation of the dyes such as 4-aminoantipyrine (4-AAP), o-dianisidine (ODA), 4-aminophenazone (APZ) etc, which are often utilized for cholesterol content estimation.⁵⁸

$$\begin{eqnarray}&&\begin{array}{c}Cholesterol\,ester+{H}_{2}O\mathop{\longrightarrow }\limits^{Cholesterol\,esterase}Cholesterol\\ \,+\,Fatty\,acid\end{array}\end{eqnarray} \tag{ 1 }$$

$$\begin{eqnarray}&&\begin{array}{c}{Cholesterol}+{O}_{2}\mathop{\longrightarrow }\limits^{{Cholesterol\; oxidase}}{Cholest}-4-\\ \,{en}-3-{one}+{H}_{2}{O}_{2}\end{array}\end{eqnarray} \tag{ 2 }$$

$$\begin{eqnarray}&&\begin{array}{c}2{H}_{2}{O}_{2}+4-Aminoantipyrine+Phenol\\ \,\mathop{\longrightarrow }\limits^{Peroxidase}\,Quioneimine\,dye+4{H}_{2}O\end{array}\end{eqnarray} \tag{ 3 }$$

Similarly, in the reaction (3) where the color products is formed, phenol can be replaced by phenolic substances, such as 2,4-dichlorophenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB), etc and aniline substances, such as N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3-methyl aniline (TOOS), N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-3,5-dimethoxy aniline (DAOS), etc.⁵⁹ Due to the conspicuous color, the chromogenic reagent 3,3,5,5-tetramethylbenzidine (TMB) is often used as a substrate for H₂O₂ detection in the presence of peroxidase to produce a blue color reaction.⁶⁰

This traditional colorimetric method has already been recommended by Chinese Society of Laboratory Medicine (CSLM).⁶¹ It shows high precision and accuracy. However, the specificity of this method may be impaired a little because cholesterol oxidase may also react with other sterols.^21,50 And some chemicals present in a sample (such as ascorbic acid and bilirubin) may consume H₂O₂. Thus, some bias during indirect cholesterol quantification may occur. Besides, it uses three-enzymes during the process, which have to be maintained bioactivities at the same time. Moreover, some inherent disadvantages such as low-stability, high-costs as well as difficult preparation largely hindered its wide application. As it is known peroxidase is needed in H₂O₂ detection.^61,62 Therefore, looking for efficient enzyme mimics is becoming an increasingly important goal in bioassays and medicine diagnostics.^60,63,64 Enzyme mimics possess many advantages over natural enzymes such as low cost, stability against denature or protease digestion.^62,65 Thus, it becomes an optimal option for enzymetic assays.

Since the emergence of artificial enzymes from 1965,⁶⁶ various materials, such as metal/metal oxide nanoparticles,^67,68 micro/nanomaterials,⁶⁶ and biomolecules.^69,70 and so on, have been extensively explored to mimic the structures and functions of natural enzymes through various approaches. In the recent decades, enzyme mimics have been applied in the detection of cholesterol.^60,62,71,72 Typically, Narsingh R. Nirala etc reported a nanomaterial, graphene quantum dots (GQDs) which can mimic the enzyme of horseradish peroxidase (HRP), for unprecedented detection of free cholesterol.⁶² Without the use of sophisticated instruments, the proposed method perceives the cholesterol using naked eye with blue compound formation. It could be made into strips. By contrasting with the color wheel, the concentration of cholesterol can be obtained. The result (Table I) showed that the proposed detection system based on GQDs allowed a wide range (0.02–0.6 mM) of cholesterol sensing with a detection limit as low as 0.006 mM along with high maximum initial velocity (V_max) and low Michaelis–Menten constant (K_m) value, suggesting that high catalytic activity, due to larger surface area of the GQDs. The research showed a great potential for enzyme mimics. But it still needs further study on the stability and detection of serum total cholesterol. What's more, the disadvantages, such as the complex preparation process, long response time and high cost, should be reminded. Yapeng Li etc prepared MXene-Ti₃C₂/CuS nanocomposites using a simple hydrothermal approach. The formation process needed to be heated at 160 ℃ for 24 h. The nanocomposites showed intrinsic peroxidase-like activity in comparison with individual MXene and CuS nanomaterials.⁶⁰ With satisfied selectivity, in the presence of COD and TMB, the MXene/CuS nanocomposites-based colorimetric sensor showed an outstanding performance for cholesterol determination with a broad linear range of 10–100 μM and a limit of detection (LOD) of 1.9 μM (Table I), which was more sensitive than many other reported methods. It showed a good linearity using serum (R² = 0.9921). However, the detective process of the colorimetric sensor in this study is closely associated with pH, temperature and H₂O₂ concentration, which required a high-standard production process for strips. Vinay Sharma and Shaikh M. Mobin demonstrated the use of a cytocompatible CuO:Graphene nanosphere (CuO:GNS) composite as a peroxidase mimic for detection of H₂O₂ and free cholesterol. The nanocomposite-based sensor presents excellent detection sensitivity for cholesterol and demonstrates a linear response (R² = 0.99) in the range of 0.1–0.8 mM with LOD as low as 78 μM. And the CuO:GNS was found to have better cytocompatibility than that of CuO only. More tests on real samples such as whole blood should be carried out for total cholesterol detection in further studies.⁷¹ On the other respect, by adding biomolecule, Ruimin Li etc developed a novel colorimetric method for detection of cholesterol with hemin-G-quadruplex DNAzyme, which can transduce the oxidation of cholesterol into the color change of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS²⁻).⁷² Oligonucleotide 5'-GTGGGTAGGGCGGGTTGG-3'(Oligo-1) can form G-quadruplex structure in the presence of K⁺. It can act as a HRP mimicking DNAzyme when binding hemin, and can catalyze the oxidation of colorless ABTS²⁻ to green ABTS^•− by H₂O₂. Even though this assay showed good linearity (R = 0.998) using serum sample, the process was complicated. At present, most of those material-based studies are still staying in the lab and require further refinements for real-life applications. So further research on whole blood sample was needed. In general, nanomaterials combined with metal/metal oxide have shown better results in cholesterol detection, which could be seen as a direction for the future. Nevertheless, preparation process remains to be simplified.

Another important aspect in colorimetric method is the supporting material selection. The initial system worth mentioning is constructed on a single sheet of paper.⁷⁶ Cholesterol from the sample reacts with CEH and COD from the subsequent pad. Later H₂O₂ formed upon this reaction passes to the measurement pad. The measurement pad is uniformly modified with aforementioned dyes. The length of the pad that changes colors upon reaction is proportional to the cholesterol concentration in the sample.⁴³ As shown in Fig. 2, firstly the sample was dropped into the hole of the test paper. After reaction, we could see the color change by reversing the paper pad.

Zoom In Zoom Out Reset image size

In addition, the materials of the paper pad itself can vary, which can have different influences for the speed and uniformity of color rendering.⁴³ To overcome these influences, Manasi Dhawane etc developed chitosan nanofiber-based cholesterol biosensor, involving colorimetric detection of the analyte.⁵¹ The uniform and bead-free chitosan nanofibers fabricated by electrospinning, were utilized for immobilizing cholesterol oxidase and peroxidase enzymes. It revealed that the intensity of color change was a reflection on cholesterol concentration. In the research, pieces of the optimized nanofibrous mat (1 × 1 sq. cm) were made to detect concentration of cholesterol. The result showed good linearity (R² = 0.99). And the nanofiber used for the pad has a high surface to volume ratio, chemical stability and alterable porosity and malleability, which are desirable properties for biomedical sensing. This kind of material is urgently needed for mass production in companies at present. For further development, more research should be done in order to broaden the detection range and lower cost etc.

Lastly, colorimetric sensors have received significant attention especially in POCT due to the rapid determination of the analytes, low-cost and wide application. The colorimetric biosensors are composed of bio-elements and biomarkers, porous membranes for support and biomarker immobilization, and a signal generating system.⁵¹ However, there are some problems in signal generating system. For example, the Accuchek Instant plus instruction card had several flaws. Most of the participants are not able to insert the test strip on their first attempt probably because the instruction card has no indicator for the correct side of the test strip to insert. After performing a finger stick the users are asked to apply a hanging drop of blood at the center of the test strip. The major problem within this test kit is the large amount of blood required for the test. Participants had to fill a well until the black circle was completely covered. Most of the participants were unable to squeeze out enough blood to complete the step. Besides, the participants must wait for 2–4 min or even longer before performing the next step. Some of the participants did not wait for enough time before proceeding to the next step, which could also lead to the detection failure.⁷⁷ In order to shorten the detection time, the research on cholesterol detection using microfluidic technology has been widely concerned in recent years. Mohammed A. Al-Rawhani etc realized the detection for total cholesterol quantification in pure blood serum by using a colorimetric complementary metal oxide semiconductor (CMOS)-based platform.⁷⁴ It could measure cholesterol concentration in human blood serum as low as 29 μM with LOD at 13 μM, which is approximately 400 times lower than average physiological level. However, the serum need be pre-treated with CEH for 10 min at 37 °C to release free cholesterol from the esterified form. And the detection range is from 29 μM to 231 μM, which is relatively narrow. After that, in 2019 Mohammed A. Al-Rawhani etc presented a versatile single CMOS chip, which integrated interleaved sensing subsystems for quadruplemode colorimetric, chemiluminescent, surface plasmon resonance and hydrogen ion measurements.⁷⁸ And it could detect glucose, cholesterol, urea and urate. Usually, to directly detect cholesterol in whole blood, a filter such as a plasma separation membrane (PSM) should be assembled because the color of the blood cells interferes with the colorimetric assay. Sungsu Park group ⁷⁹ and Fang Li group ⁷⁵ developed PSM-integrated 3D-paper-based microfluidic analytical devices (μPADs) and double-layered 3D-μPADs separately. As demonstrated in the research, the 3D-μPADs could detected multiple substances simultaneously. As shown in Table I, the detection range of cholesterol is 0.01–0.4 mmol l⁻¹, and the detection limits was 0.07 μmol/L. The detection time is short (70 s). As a direction, more attention should be paid to the connecting issue between detection strips and signal generating system and the interference of the blood cells in the following study.

To eliminate the effects of ambient light, researches on signal generating system are proposed. For example, magnetic nanoparticles-based optical detection system depends on the aggregation of initially dispersed particles to yield a straightforward color change from yellow to brown,⁸ which is easily to form uniform color products. Furthermore, a method of smartphone-based POCT urinalysis under various conditions of illumination was proposed,⁹ which is convenient and adaptable to social trends. These methods could be considered in detection other samples, such as serum total cholesterol.

Electrochemical method is the most frequently applied in the fabrication of enzyme-based cholesterol biosensors. A POCT device with an electrochemical sensor exhibits high precision, fast response, low cost, and portability.^3,80–84

Based on the different enzymes used, it can be classified as two methods: cholesterol oxidase method ^85,86 and cholesterol dehydrogenase method.^87,88 The basic principles of the reaction process are also introduced as follows (Figs. 3a and 3b).

Zoom In Zoom Out Reset image size

As shown in Fig. 3b, to avoid the interference of reducing substances in blood (such as ascorbic acid etc) in cholesterol oxidase method, Yuzo Kayamori etc developed an endpoint assay for serum cholesterol, based on a CEH-cholesterol dehydrogenase (CDH)-ultraviolet method. However, there was a disadvantage at equilibrium, in which the CDH reaction gave incomplete conversion of cholesterol to cholest-4-ene-3-one (Fig. 3b). To overcome this disadvantage, hydrazine monohydrate, which is poisonous, should be added to the reaction mixture to remove cholest-4-ene-3-one, which allows the reaction to proceed to completion and gives stoichiometric production of β-NADH from the reaction of β-NAD⁺ with cholesterol. So the CEH-COD-peroxidase chromogenic method is still widely used.

Apart from the enzyme-based classification, the electrochemical detection methods for cholesterol detection can be also divided based on different reaction principles: the consumption of oxygen and the rate of H₂O₂ formation by an enzyme reaction. Although the former is not disturbed by bilirubin and ascorbic acid, H₂O₂ is easier to transfer electrons. So the detection of cholesterol content is mostly reported by detecting the rate of H₂O₂ formation in recent years.⁸⁹

Another classification method based on the recording techniques: the amperometric method and the voltammetric method. The amperometric total cholesterol measurement is performed based on the reactions (1, 2, 4) that occur on the surface of the working electrode.

$$\begin{eqnarray}&&{H}_{2}{O}_{2}\mathop{\longrightarrow }\limits^{Electrocatalytic\,oxidation}{O}_{2}+2{H}^{+}+2{e}^{-}\end{eqnarray} \tag{ 4 }$$

Reaction (4) produces an electric output current that can be recorded via the computer-controlled data acquisition software.⁹⁰

Ksenia V. Derina etc used voltammetric sensor to detect total cholesterol. In their study it suggests that voltammetry, as an analytical technique, presents higher accuracy than the amperometric methods.⁴⁶ They fabricated a novel sensor by co-immobilizing COD and HRP on the porous graphite surface. It exhibits certain selectivity for the analyte in the presence of other compounds present in the sample including ascorbic acid, glucose, lactic acid, and uric acid. To evaluate the performance of voltammetric method, more studies should be carried out and further results should be obtained. At present the amperometric method is more widely used due to its convenience. The following are various studies based on amperometric method.

For the electrochemical sensors, the electrodes are usually fabricated using screen-printing technique. The typical electrodes are shown in Fig. 4a.⁹¹ It is a two-electrode system, including a working electrode and a reference electrode. The electrodes are fabricated on the substrates, and then the reagents are immobilized in the reaction area. With the increasing demand for higher sensitivity and selectivity, three-electrode system and four-electrode system have been provided. The supporting or the counter electrode is added in the three-electrode system (Fig. 4b).⁸⁶ And hematocrit correction electrode is added in the four-electrode system in extra (Fig. 4c). The blood is inhaled into the test paper by siphon and passes through the conductance measurement area. The conductance test electrodes are composed of the trigger electrode and the second pair of electrodes measuring the hematocrit of blood flowing through the conductance measurement area. Then the blood enters the electrochemical measurement reaction chamber for total cholesterol determination. The measured value of total cholesterol is calibrated by using the hematocrit (Hct) value, based on the influence curve of Hct on blood glucose concentration.⁹² Finally, the value is recorded and displayed on the tester.

Zoom In Zoom Out Reset image size

For detecting systems, it should be considered in terms of sensitivity, LOD, fast response time, etc The initial electrochemical method for detection cholesterol requires high voltage to produce an electric current.⁹³ However, as the biological fluids such as serum contains high concentrations of other analytes, protein, etc, it is desirable to develop a sensing platform which can be operated at low voltages so as to prevent ChOx simultaneously reacting with other electroactive analytes in the samples, for example, uric acid, ascorbic acid, bilirubin, pyruvate, glutathione and produce false positive signals as well. That is why the second-generation enzymatic biosensor is widely used, which requires low voltages owing to the involvement of the electron mediator.⁴⁶

To minimize the effect of interferences, techniques have been developed using the redox mediator ferricyanide to reduce the reaction potential of hydrogen peroxide. In order to avoid unnecessary oxidation of other electroactive analytes and obtain high sensitivity, the working potential of 300 mV is appropriately used.⁹¹ In recent years, to further improve the sensitivity of those biosensors, some cholesterol electrochemical biosensor researches are focusing on the direct electron transfer between ChOx and electrode surfaces.^46,94–99 Sheela Berchmans' group and Shelley D. Minteer's group used Ni(OH)₂/NiOOH¹⁰⁰ and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, free radical)¹⁰¹ as redox catalysts to mediate electron transfer respectively. They both showed excellent catalytic properties toward hydrogen peroxide. And the former one possessed long term stability and wide linear range, as shown in Table II. TEMPO, as an organic redox catalysts, could be used to monitor H₂O₂ in broad pH range (≥4) at 530 mV, which was beneficial to specially detect cholesterol and could be used for various populations with different pH in blood. Due to the high potential required, Nidhi Chauhan etc use pencil graphite electrode (PGE) bounded cholesterol oxidase as working electrode, silver/silver chloride (Ag/AgCl) as reference electrode and Cu wire as auxiliary electrode. It has the advantage over previous amperometric methods that it requires low potential to generate electrons from H₂O₂, which does not induce ionization of serum substances.⁴⁷ COD is immobilized on PGE through chemisorption, a weak Van der Waals forces. Moreover, the current value is well correlated (R = 0.99) with the serum cholesterol concentration by standard enzymatic colorimetric method. The detection range was wide, which could be seen in Table II. Besides, no interference by metabolites was observed in the method. It may provide a path for the study of for the anti-interference effects using the serum or blood.

On the other hand, in theory, negative potential detection method is put forward for its little interference and the benefit for the human body. So it becomes another trend for the researchers. Pedro Salazar etc described a situ electrodeposition of ChOx-modified polydopamine (PDA) thin film on nanostructured-screen printed electrodes (SPEs).¹⁰² The applied potential was fixed to −0.1 V against the internal Ag pseudo-reference electrode. According to the reaction 1, 2 and 5, electrical current was produced.

$$\begin{eqnarray}&&\begin{array}{c}\left[PW\right]F{e}_{4}{K}_{4}{\left(Fe{\left(CN\right)}_{6}\right)}_{3}+2{H}_{2}O\to \left[PB\right]F{e}_{4}{K}_{4}{\left(Fe{\left(CN\right)}_{6}\right)}_{3}\\ \,+\,4O{H}^{-}+4{K}^{+}\end{array}\end{eqnarray} \tag{ 5 }$$

It showed a rapid response after cholesterol addition, reaching the stationary current after ca. 7 s. The interfering substances, including ascorbic acid, glucose, acetaminophen, uric acid, fructose, sucrose and lactose, produced negligible effects on the actual cholesterol current, thus confirming the high selectivity for cholesterol detection. Prussian Blue (PB) and multi-wall carbon nanotubes (MWCNTs) were used to form a SPE/PB/MWCNTox/PDA@ChOx electrode as working electrode, and silver as pseudo-reference and counter electrode. Totally, compared with other electrodes,^103–106 it showed rapid response time, good anti-interference performance, high sensitivity. To be fair it has relatively narrow detection range (Table II). The preparation process should also be further simplified.

In order to optimize the detecting system, the immobilization for the enzyme is another important part that worth exploring. Since the activity of enzymes is closely related with pressure, temperature and pH, the immobilization of enzyme is crucial in order to maintain its activity.

Immobilization methods can be classified based on different standards. From the aspects of support, it can be divided into support-requiring and support-free. According to the nature of linkage between enzyme and the supporting material, it can be also classified into categories such as adsorption-based, covalent binding and cross-linking, entrapment and encapsulation.⁴³ The adsorption method (Fig. 5a) is simple, inexpensive and requires no chemical modification of enzymes. Unfortunately, this type of immobilization can result in subsequent leaching. At the same time, the distribution of the enzyme in paper matrix is difficult to control.¹⁰⁷ Besides, this method is also highly influenced by the reaction/deposition conditions, like pH that will impact the surface charge. Covalent binding (Fig. 5b) rigidifies structure of the enzyme.¹⁰⁸ If the enzyme is deactivated, the support, sometimes costly, is also rendered useless. In the case of entrapment (Fig. 5c) enzymes are typically enclosed in a polymer matrix (organic polymer, sol-gel, which is yellow in Fig. 5c). In this case the substrate is consecutively exposed to polymer solutions of the opposite charge. Immobilization of enzyme is achieved by electrostatic interactions or hydrogen bonds in some cases.¹⁰⁹ Encapsulation (Fig. 5d) results in similar characteristics to entrapment. In this case, the immobilization could be carried independently in an optimized environment.⁴³ And many engineered enzymes with cellulose binding domains have already been commercially available, but the price was still high.^104,110

Zoom In Zoom Out Reset image size

Huseyin Bekir Yildiz etc designed two types of amperometric cholesterol biosensors by physically entrapping cholesterol oxidase in conducting polymers, thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-cop-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). The results showed that ChOx immobilized in PEO-co-PPy displayed higher affinity towards the substrate.¹⁰⁴ But it required high potential (0.7 V). M. Alagappan etc added conductive material (K₄[Fe(CN)₆) to lower the potential. They immobilized the ChOx on gold nanoparticles-functionalized-MWCNT-PPy nanocomposite modified electrode. PPy acts as a support matrix to hold ChOx and the presence of Au-f-MWCNT increases the electrical conductivity. However, the positive potential couldn't prevent the interference reaction.¹⁰⁶ Saniye Soylemez etc designed a simple and robust cholesterol biosensor by immobilizing ChOx onto a conducting polymer modified graphite electrode. Monomer, (Z)-4-(4-(9H-carbazol9-yl) benzylidene)-2-(4-nitrophenyl) oxazol-5(4H)-one (CBNP) was synthesized. After electropolymerization, it was used to immobilize the enzyme. As shown in Table II, it still had some disadvantages, such as narrow detection range, poor stability, complex process and so on.¹⁰⁵

Based on the analysis of the above two methods, detection system has also been advanced to be disposable together with the test strip. However, this kind of one-component integration of electrochemical devices is still not available for a reasonable price. Instead, colorimetric system can realize equipment-free quantitative detection, which can be accomplished by appropriate design of the device. For example, cell phone cameras and downloadable software could also be used by simply installing appropriate softwares for color-intensity-based quantification. From this respect, colorimetric method is superior to electrochemical method. In summary, for low-cost applications in real-life situations, colorimetric or electrochemical system should be further improved to own the properties including self-contained and one-component design, using unprocessed sample, no specialized training required and easy to interpret.⁴³

In conclusion, the key point of the cholesterol detection is to obtain the methods with low cost, simple preparation process and easy mass production under the premise of high sensitivity, good linearity, strong anti-interference ability, good stability, wide detection range and short response time.