Application of Peroxidase-Mimic Mn2BPMP Boosted by ADP to Enzyme Cascade Assay for Glucose and Cholesterol

Abstract

The Mn₂BPMP complex has an intrinsic peroxidase-like activity in the pH range of 5 to 8, especially a maximum activity at pH 7, while most peroxidase mimics operate at an acidic pH (mainly pH 4). Its peroxidase-like activity is high among small-molecule-based peroxidase mimics with a high reproducibility. In addition, we recently revealed that adenosine mono/diphosphate (AMP and ADP) significantly boosted the peroxidase-like activity of Mn₂BPMP. These advantages imply that Mn₂BPMP is suitable for biosensing as a substitute for horseradish peroxidase (HRP). Herein, we established a colorimetric one-pot assay system using the enzyme cascade reaction between analyte oxidase and ADP-boosted Mn₂BPMP. The simple addition of ADP to the Mn₂BPMP-based assay system caused a greater increase in absorbance for the same concentration of H₂O₂, which resulted in a higher sensitivity. It was applied to one-pot detection of glucose and cholesterol at 25 °C and pH 7.0 for a few minutes.

1. Introduction

Horseradish peroxidase (HRP) is a natural metalloenzyme that catalyzes the oxidation of various optical organic substrates in the presence of hydrogen peroxide (H₂O₂). That is, peroxidase activity causes quantitative optical signal changes such as color and fluorescence in proportion to the concentration of H₂O₂. Due to its high specificity and efficiency, HRP has been widely utilized in biosensors for the detection of various biomolecules such as glucose and antigens through enzyme cascade assays [1,2,3], immunoassays [4,5,6], and aptasensors [7,8,9]. However, HRP has intrinsic drawbacks, such as vulnerability of the catalytic activity to environmental conditions and the high cost of preparation, purification, and storage processes. These disadvantages limit its further practical applications.

Various peroxidase mimics have emerged as an alternative to HRP, such as magnetic nanoparticles [10,11], gold nanoparticles [12,13], metal-organic frameworks [14,15,16], graphene oxides [17,18], and small molecules [19,20]. However, most of the reported peroxidase mimics still suffer from the requirement of high temperature and acidic conditions for their peroxidase-like activities, and from poor reproducibility due to the batch-to-batch variation of nanomaterials [21]. Furthermore, most of them demand relatively long response times and multistep processes for biosensing. Therefore, the development of a peroxidase mimic that overcomes these disadvantages is highly desirable for the practical and simple detection of various biomolecules.

Recently, our group reported that Mn₂BPMP (two Mn²⁺ ions-coordinated BPMP; 2,6-bis[(bis(2-pyridylmethyl)amino)-methyl]-4-methylphenolate) possesses a peroxidase-like activity [22]. This was demonstrated using a colorimetric assay with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the chromogenic peroxidase substrate and H₂O₂. Notably, Mn₂BPMP works well at a neutral pH (pH 7.0) and responds quickly to ABTS and H₂O₂ within a minute. Moreover, the batch-to-batch variation is irrelevant in the use of Mn₂BPMP. Based on the peroxidase-like activity of Mn₂BPMP, H₂O₂ can be quantitatively detected by the Mn₂BPMP and ABTS system. This suggests that Mn₂BPMP can be further utilized for the indirect detection of various biomolecules engaged in reactions generating H₂O₂. Recently, it was revealed that adenosine mono/diphosphate (AMP and ADP) could boost the peroxidase-like activity of Mn₂BPMP, lowering the Michaelis−Menten constant (K_M) toward H₂O₂ and ABTS [23]. A high peroxidase-like activity is necessary for peroxidase mimics for the sensitive detection of biomolecules in the application. In addition, the Kong group reported that adenosine di/triphosphate (ADP and ATP) not only enhanced the peroxidase-like activity of peroxidase mimic, G-quadruplex-hemin DNAzyme, but also stabilized the bluish-green ABTS^•+, which is unstable and decays to a colorless compound [24,25]. Therefore, we recruited them for the rapid and sensitive detection of biomolecules based on enzyme cascade reactions.

Herein, we constructed an Mn₂BPMP/ADP/ABTS/oxidase system for the one-pot colorimetric assay of biomolecules by coupling two cascade reactions: oxidation of the target analyte by related analyte oxidase (AOx) and oxidation of ABTS by generated H₂O₂ and ADP-boosted Mn₂BPMP (Scheme 1). As the oxidized ABTS radical has a bluish-green color, the color of the assay solution became darker in proportion to the concentration of the analyte. The enhanced peroxidase-like activity of Mn₂BPMP by ADP caused a greater change in absorbance for the same concentration of analyte than in the assay system without ADP. In addition, ADP, a stabilizer of ABTS^•+, maintained a darker bluish-green color for the assay solution than without ADP, making it possible to distinguish the analyte concentration more clearly with the naked eye. This system was successfully applied to the one-pot detection of glucose, which is related to human diseases such as diabetes mellitus and hyperlipidemia, and has great importance in clinical diagnosis [26]. In addition, it was extended to the detection of cholesterol, with great potential for further applications.

2. Materials and Methods

2.1. Materials and Instrumentation

2,6-Bis(hydroxymethyl)-p-cresol, manganese acetate tetrahydrate (Mn(OAc)₂·4H₂O), sodium perchlorate (NaClO₄), cholesterol bioreagent, cholesterol oxidase from microorganisms, D-(-)-fructose, D-(+)-glucose, glucose oxidase from Aspergillus niger, D-(+)-maltose monohydrate, adenosine 5′-triphosphate disodium salt hydrate (ATP), adenosine 5′-monophosphate monohydrate (AMP), and Triton X-100 were purchased from Sigma-Aldrich, Seoul, Korea. Adenosine 5′-diphosphate (ADP) disodium salt hydrate, 2,2′-dipicolylamine, hydrogen peroxide, and D-(+)-sucrose were purchased from Tokyo Chemical Industry, Seoul, Korea. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS) was purchased from Alfa Aesar, Seoul, Korea. Thionyl chloride and triethylamine were purchased from Daejung Chemical Industry, Gyeongbuk, Korea. Sodium acetate trihydrate (NaOAc·3H₂O) was purchased from Junsei Chemical Industry, Seoul, Korea. D-(+)-Galactose was purchased from Janssen Pharmaceuticals, Seoul, Korea. Lactose monohydrate was purchased from Samchun Chemicals, Seoul, Korea. All of the chemicals were used without further purification.

The optical density spectrum was recorded on a BioTek Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) using a 96 well plate. Absorbance spectra were recorded on an Agilent Cary 8454 UV−VIS spectrophotometer (Agilent, Santa Clara, CA, USA) using a 1 cm path length quartz cell. ¹H-NMR and ¹³C-NMR spectra were recorded on a JEOL (400 MHz) NMR spectrometer (JEOL, Tokyo, Japan). Melting points were measured using a BUCHI melting point M-565 apparatus (Büchi, Flawil, Switzerland). The mass spectrum was recorded on an Agilent 6520 accurate-mass quadrupole time-of-flight mass spectrometer (MS) (Agilent, Santa Clara, CA, USA) with an electrospray ionization (ESI) source.

2.2. Effect of Adenosine Phosphates on the Peroxidase-like Activity of Mn₂BPMP

Various adenosine phosphates (ATP, ADP, and AMP; 2 μM) were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (1 μM) and ABTS (1 mM). Then, H₂O₂ (5 mM) was added. The optical density spectra for these samples were recorded at 10-s intervals for 5 min at 420 nm and 25.0 °C.

2.3. Effect of ADP on the Peroxidase-like Activity of Mn₂BPMP

Various concentrations of ADP were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (1 μM) and ABTS (1 mM). Then, H₂O₂ (5 mM) was added. The UV−VIS spectra for these samples were recorded at 10-s intervals for 3 min at 420 nm and 25.0 °C.

2.4. Colorimetric Detection of H₂O₂ in the Presence or Absence of ADP

Various concentrations of H₂O₂ were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (1 μM) and ABTS (1 mM) in the presence or absence of ADP (2 μM). The UV−VIS spectra for these samples were recorded at 10-s intervals for 3 min at 420 nm and 25.0 °C.

2.5. Colorimetric Detection of Glucose and Selectivity/Interference Experiments in the Presence or Absence of ADP

Various concentrations of glucose were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (2 μM), ABTS (1 mM), and glucose oxidase (GOx) (1 U/mL) in the presence or absence of ADP (4 μM). The UV−VIS spectra for these samples were recorded at 30-s intervals for 15 min at 420 nm and 25.0 °C.

For the selectivity and interference experiments, various glucose analogs (7 mM), such as fructose, galactose, lactose, sucrose, and maltose, were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (2 μM), ABTS (1 mM), ADP (4 μM), and GOx (1 U/mL) in the presence or absence of glucose (700 μM). After 7 min, the UV−VIS spectra for these samples were recorded at 420 nm and 25.0 °C.

2.6. Colorimetric Detection of Glucose in Human Serum

Human serum was prepared by ultrafiltration and the glucose concentration of the human serum was measured using a glucose meter. The 10-fold diluted serum samples, which were spiked with various concentrations of glucose (5, 10, and 25 mM), were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (2 μM), ABTS (1 mM), glucose oxidase (GOx) (1 U/mL), and ADP (4 μM). The UV−VIS spectra for these samples were recorded at 7 min at 420 nm and 25.0 °C.

2.7. Colorimetric Detection of Cholesterol in the Presence or Absence of ADP

To prepare a 10 mM stock solution of cholesterol, 38.6 mg of cholesterol was first dissolved in a mixture of 1 mL of Triton X-100 and 0.5 mL of isopropyl alcohol in a warm water bath at 60 °C. Then, 8.5 mL of 200 mM of Tris pH 7.0 buffer solution was added to give a stock solution of cholesterol. It was diluted using 200 mM of Tris pH 7.0 buffer solution for working solutions.

Various concentrations of cholesterol were added to a buffer solution (Tris, pH 7.0, 20 mM, H₂O to acetonitrile v/v = 19:1) containing Mn₂BPMP (2 μM), ABTS (1 mM), and cholesterol oxidase (ChOx) (1 U/mL) in the presence or absence of ADP (4 μM). The UV−VIS spectra for these samples were recorded at 30-s intervals for 5 min at 420 nm and 25.0 °C.

3. Results and Discussion

3.1. Effect of ADP on the Peroxidase-like Activity of Mn₂BPMP

Mn₂BPMP was synthesized as the pale-yellow crystals in three steps: chlorination of 2,6-bis(hydroxymethyl)-p-cresol with thionyl chloride, nucleophilic substitution with 2,2′-dipicolylamine to produce H-BPMP, and H-BPMP chelation with two Mn²⁺ ions. The synthesized H-BPMP and Mn₂BPMP were confirmed by ¹H and ¹³C NMR spectra and mass spectrum, respectively (Figures S1–S4). Mn₂BPMP is a small molecule-based peroxidase mimic with an intrinsic peroxidase-like activity at pH 5–8, especially at pH 7 [22]. Mn₂BPMP oxidizes colorless ABTS to bluish-green ABTS^•+ using H₂O₂, and the concentration of H₂O₂ can be quantified through the color change. In this study, it was first confirmed that H₂O₂ of sub 100 μM could be detected within 1 min using only 1μM of Mn₂BPMP as a control (Figure S8).

The effect of adenosine phosphates (ATP, ADP, and AMP) as the additive booster on the peroxidase-like activity of Mn₂BPMP was confirmed to enhance the sensitivity for the detection of H₂O₂. Adenosine phosphates corresponding to two equivalents of Mn₂BPMP were added to the Mn₂BPMP/ABTS/H₂O₂ system in a pH 7.0 Tris buffer solution. In the case of ATP, the increase in absorbance versus time occurred more slowly than the control assay solution without adenosine phosphates, whereas in the case of ADP and AMP, the absorbance of the assay solution increased more rapidly with time than the control (Figure 1a). The assay solution containing ADP not only increased the absorbance approximately six times compared to the control at 1 min, but also showed a significantly dark bluish-green color compared to the other assay solutions (Figure 1b). This pronounced color difference was presumably due to the two beneficial functions of ADP: (1) enhancement of peroxidase-like activity of Mn₂BPMP and (2) stabilization of ABTS^•+. In particular, compared to other conditions, the assay solution containing the ADP showed a remarkably rapid increase in absorbance versus time at the beginning of catalytic reaction, indicating that Mn₂BPMP used H₂O₂ with the help of ADP to rapidly oxidize ABTS. This result suggests that ADP plays a crucial role in greatly enhancing the peroxidase-like activity of Mn₂BPMP. Unstable ABTS^•+ usually is decayed or over-oxidized to form a colorless compound over time, which gradually loses the bluish-green color of the assay solution. For this reason, bluish-green ABTS^•+ with a molar extinction coefficient of 36,000 M⁻¹ cm⁻¹ at 420 nm are present in a very small proportion compared to other stable ABTS analogues in the ABTS-based assay systems [24,25,27]. Interestingly, in the Mn₂BPMP/ABTS/H₂O₂ system, it is speculated that ADP stabilized ABTS^•+, allowing for a greater proportion of ABTS^•+ to be present in the assay solution.

In this study, ADP was recruited as the additive booster of the peroxidase-like activity of Mn₂BPMP and the stabilizer of ABTS^•+ in order to improve the detection sensitivity for H₂O₂, resulting in a high sensitivity in the enzyme cascade assay system for the detection of various analytes. The optimization concentration of ADP as the additive booster was confirmed in the Mn₂BPMP/ABTS/H₂O₂ system. As shown in Figure 2 and Figure S5, the absorbance at 420 nm dramatically increased with the concentration of ADP and reached a plateau at 2 μM within 1 min. Hence, subsequent experiments were conducted by adding ADP corresponding to twice the concentration of Mn₂BPMP in Mn₂BPMP-based colorimetric assay systems.

Colorimetric detection of H₂O₂ was conducted in the presence or absence of 2 μM of ADP to compare the effect of ADP. As shown in Figure 3 and Figures S6 and S7, the absorption signal was notably increased when ADP was present, and it was also observed with the naked eye. For H₂O₂, the linear range was 100 to 1000 μM, and the limit of detection (LOD) (S/N = 3) was 9.7 μM with R² = 0.997 in the presence of ADP, and the linear range was 100 to 1000 μM and the LOD (S/N = 3) was 85 μM with R² = 0.983 in the absence of ADP (Figure S8). Notably, a difference in the color of the assay solution was evident even at a low concentration of H₂O₂ in the presence of ADP. In addition, the LOD was decreased by about 9-fold when ADP was added, indicating that ADP could enhance the sensitivity for H₂O₂ in the Mn₂BPMP/ABTS/H₂O₂ system. As this enhanced sensitivity enabled the detection of lower concentrations of analytes in a shorter time, the ADP-boosted Mn₂BPMP/ABTS/H₂O₂ system would be more suitable for application in enzyme cascade assays.

3.2. Colorimetric Detection of Glucose Using the ADP/Mn₂BPMP/ABTS/GOx System

Next, based on the ADP-boosted peroxidase-like activity of Mn₂BPMP, colorimetric detection of glucose was conducted under an ADP/Mn₂BPMP/ABTS/GOx system. One equivalent of glucose was oxidized by GOx generating one equivalent of H₂O₂, as shown in Equation (1):

(1)$\mathsf{\beta }-\mathrm{D}-\mathrm{glucose} +{ \mathrm{O}}_2 \stackrel{\mathrm{GOx}}{\to } \mathrm{D}-\mathrm{glucono}-1,5-\mathrm{lactone}+{\mathrm{H}}_2{\mathrm{O}}_2$

Therefore, a quantitative analysis of H₂O₂ enables an indirect quantitative analysis of glucose. In detail, glucose was oxidized by GOx, which generated H₂O₂ as a byproduct. Subsequently, with the help of the generated H₂O₂, the enhanced peroxidase-like activity of Mn₂BPMP by ADP catalyzed the oxidation of ABTS to ABTS^•+ to give a bluish-green color in the solution. As higher concentrations of ABTS^•+ gave a deeper bluish-green color, a quantitative analysis of glucose was enabled via measuring the absorbance by UV−VIS spectroscopy and naked eye observation. Scheme 1 illustrates the principle of the biosensing of the Mn₂BPMP/ABTS/GOx system based on these cascade reactions.

Various concentrations of glucose were added to the Mn₂BPMP/ABTS/GOx system in a Tris buffer (pH 7.0) solution in the presence or absence of ADP. The absorbance at 420 nm increased with an increasing concentration of glucose (Figure 4a and Figures S9 and S10). For glucose, the linear range was 100 to 700 μM and the LOD (S/N = 3) was 16 μM with R² = 0.996 in the presence of ADP, and the linear range was 100 to 500 μM and the LOD (S/N = 3) was 59 μM in the absence of ADP with R² = 0.982 (Figure S11). Due to the highly enhanced peroxidase-like activity of Mn₂BPMP by ADP, not only was the color change clearly observed by the naked eye (Figure 4b), but it also lowered the LOD for glucose. In previous literature, most enzyme cascade-based glucose assay systems using nanomaterial-based peroxidase mimics such as ferromagnetic nanoparticles and gold nanoparticles were a method of pre-incubating glucose and glucose oxidase, lowering the pH, and detecting H₂O₂ generated by using peroxidase mimics with a low working pH, as shown in Table S1 [10,11]. Due to this, there are disadvantages, in that it is inconvenient to go through a two-step process and it takes tens of minutes. On the other hand, our system showed that it was possible to quantitatively detect a glucose concentration in 7 min in one pot. Moreover, our system is cost-effective and stable compared with the use of HRP, owing to the characteristics of a small-molecule-based peroxidase mimic.

To confirm that the ADP/Mn₂BPMP/ABTS/GOx system has a high selectivity toward glucose and there was no interference by glucose analogues, a 10-fold higher concentration of fructose (Fru), galactose (Gal), lactose (Lac), maltose (Mal), and sucrose (Suc) was added to the system, individually, in the absence or presence of glucose. Then, the absorbance at 420 nm was measured after 7 min by UV−VIS spectroscopy. As shown in Figure 5a, no significant signals in absorbance at 420 nm were observed without glucose and there was no interference effect by the glucose analogues with glucose. It was also observed with the naked eye, as shown in Figure 5b. These results demonstrate that the proposed system can efficiently and selectively detect glucose, even with the naked eye. More importantly, real-time detection of glucose is possible, as this system does not require any multistep processes or long response time. Furthermore, the applicability of the ADP/Mn₂BPMP/ABTS/GOx system for the detection of glucose in human serum was verified. Human serum was prepared by ultrafiltration to remove yellowness, and the glucose concentration of the filtered human serum was measured to be 5.35 ± 0.02 mM using the glucose meter. The serum samples spiked with known glucose concentrations (5, 10, and 25 mM) were added to the ADP/Mn₂BPMP/ABTS/GOx system, and the glucose concentration was detected by measuring the absorbance. Recovery of the glucose concentrations in the serum samples ranged from 82.79% to 100.16%, with the relative standard deviation (RSD) ranging from 5.26% to 9.12% (Table S2). It was shown that glucose can be quantitatively detected at concentrations much higher than normal in serum samples, suggesting the possibility that the ADP/Mn₂BPMP/ABTS/GOx can be utilized in the diagnosis of diseases related to high glucose levels.

3.3. Colorimetric Detection of Cholesterol Using the ADP/Mn₂BPMP/ABTS/ChOx System

To confirm whether the ADP/Mn₂BPMP/ABTS/oxidase system could be extended to the general platform for the colorimetric detection of other biomolecules, the detection of cholesterol was also conducted by simply substituting ChOx for GOx. As cholesterol, the precursor of steroid hormones and bile acids, is associated with many human diseases, such as arteriosclerosis, hypertension, hypothyroidism, and hypocholesterolemia [28,29], fast and reliable detection of cholesterol is highly important in clinical diagnosis. One equivalent of cholesterol is oxidized by ChOx generating one equivalent of H₂O₂, as shown in Equation (2):

(2)$\mathrm{cholesterol} +{ \mathrm{O}}_2 \stackrel{\mathrm{ChOx}}{\to } \mathrm{cholest}-4-\mathrm{en}-3-\mathrm{one}+{\mathrm{H}}_2{\mathrm{O}}_2$

Therefore, a quantitative analysis of cholesterol could be conducted in a similar way to the detection of glucose.

Various concentrations of cholesterol were added to the Mn₂BPMP/ABTS/ChOx system in the Tris buffer (pH 7.0) solution in the presence or absence of ADP. The absorbance at 420 nm increased with an increasing concentration of cholesterol and the color change could be noticed with the naked eye in the presence of ADP (Figure 6 and Figures S12 and S13). For cholesterol, the linear range was 20 to 150 μM and the LOD was 4.8 μM with R² = 0.994 in the presence of ADP, and the linear range was 20 to 100 μM and the LOD was 12 μM with R² = 0.986 in the absence of ADP (Figure S14). The proposed ADP/Mn₂BPMP/ABTS/oxidase system could be applied for the detection of cholesterol as well. We envision that our system can be further utilized as a general platform in colorimetric biosensors for the real-time detection of various biomolecules accompanied by the generation of H₂O₂.

4. Conclusions

In this study, we demonstrated that the ADP/Mn₂BPMP/ABTS/GOx system can be efficiently applied to the colorimetric detection of glucose based on the highly enhanced peroxidase-like activity of Mn₂BPMP by ADP with the ability to stabilize ABTS^•+. In this system, the linear range was 100–700 μM and LOD was 16 μM for glucose. In particular, because of the highly increased signals due to the involvement of ADP, glucose can be easily detected even with the naked eye, which is considerably advantageous in the detection of biomolecules. As this system does not require a long response time or any multistep processes, the real-time detection of glucose was successfully demonstrated within a few minutes in a simple way. As an extension, we also demonstrated that the colorimetric detection of cholesterol was enabled based on our system simply replacing GOx with ChOx. Therefore, we anticipate that the proposed ADP/Mn₂BPMP/ABTS/oxidase systems can be further developed into a general platform for the real-time detection of various biomolecules. Furthermore, as a peroxidase mimic, Mn₂BPMP may be a practical alternative to the natural enzyme HRP in a variety of fields, such as biosensors.

Author Contributions

Conceptualization, M.S.H.; funding acquisition, M.S.H.; investigation, N.L. and S.Y.; methodology, N.L. and S.Y.; project administration, M.S.H.; resources, N.L. and Y.L.; supervision, M.S.H.; validation, S.Y.; visualization, N.L. and S.Y.; writing—original draft, N.L. and S.Y.; writing—review and editing, S.Y. and M.S.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT), grant number NRF-2020R1A2B5B01002392.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Scheme

Scheme 1. Schematic illustration of the Mn2BPMP/ADP/ABTS/AOx system.

Figure 1. (a) Changes in the optical density and (b) photograph of the Mn2BPMP/ABTS/H2O2 system in the presence of various adenosine phosphates (ATP, ADP, and AMP). Adenosine phosphates = 2 µM; Mn2BPMP = 1 µM; ABTS = 1 mM; H2O2 = 5 mM in a buffer solution (Tris, 20 mM, pH 7.0).

Figure 2. Changes in the absorbance of the Mn2BPMP/ABTS/H2O2 system in the presence of various concentrations of ADP (from 0 to 3 µM) after 3 min. Inset: The plot of the absorbance at 420 nm versus the concentration of ADP (from 0 to 3 µM) after 3 min. Mn2BPMP = 1 µM; ABTS = 1 mM; H2O2 = 5 mM in a buffer solution (Tris, 20 mM, pH 7.0).

Figure 3. (a) The plot of the absorbance at 420 nm and (b) the photograph versus the concentration of H2O2 (0 to 5 mM) for the Mn2BPMP/ABTS system in the presence or absence of ADP (2 µM) after 1 min. Mn2BPMP = 1 µM; ABTS = 1 mM in a buffer solution (Tris, 20 mM, pH 7.0).

Figure 4. (a) The plot of the absorbance at 420 nm and (b) photograph versus the concentration of glucose (0 to 2 mM) for the Mn2BPMP/ABTS/GOx system in the presence or absence of ADP (4 µM) after 1 min. Mn2BPMP = 2 µM; ABTS = 1 mM; GOx = 1 U/mL in a buffer solution (Tris, 20 mM, pH 7.0).

Figure 5. (a) The absorbance at 420 nm and (b) photograph of the Mn2BPMP/ADP/ABTS/GOx system with various glucose analogs (7 mM) in the presence or absence of glucose (700 µM) after 7 min. Mn2BPMP = 2 µM; ADP = 4 µM; ABTS = 1 mM; GOx = 1 U/mL in a buffer solution (Tris, 20 mM, pH 7.0).

Figure 6. (a) The plot of the absorbance at 420 nm and (b) photograph versus the concentration of cholesterol (0 to 300 µM) for the Mn2BPMP/ABTS/ChOx system in the presence or absence of ADP (4 µM) after 1 min. Mn2BPMP = 2 µM; ABTS = 1 mM; ChOx = 1 U/mL in a buffer solution (Tris, 20 mM, pH 7.0).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/chemosensors10020089/s1. Figures S1–S3: ¹H and ¹³C NMR spectra of the synthesized compounds, Figure S4: Mass spectrum of Mn₂BPMP, Figure S5: Optimization of ADP concentration in the Mn₂BPMP/ABTS/H₂O₂ system, Figures S6–S8: H₂O₂ titration using the Mn₂BPMP/ABTS/H₂O₂ system in the presence or absence of ADP, Figures S9–S11: Glucose titration using the Mn₂BPMP/ABTS/GOx system in the presence or absence of ADP, Figures S12–S14: Cholesterol titration using the Mn₂BPMP/ABTS/ChOx system in the presence or absence of ADP.

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