A novel probe based on phenylboronic acid functionalized carbon nanotubes for ultrasensitive carbohydrate determination in biofluids and semi-solid biotissues

An ultrasensitive SPME probe based on phenylboronic acid functionalized CNTs is applied for direct in vitro or in vivo recognition of carbohydrates in biofluids as well as semi-solid biotissues.


Introduction
Carbohydrates are known to be involved in a wide range of biological processes. 1 Simultaneously, the concentration of carbohydrates in biological system is vital to several pathological processes. For example, diabetes mellitus, which is one of the biggest public health threats, demands continuous carbohydrate monitoring. 2 Thus, precise determination of carbohydrates is necessary for not only fundamental researches but also clinical diagnoses.
Unlike nucleic acids, amino acids and lipids, determination of carbohydrates in aqueous solution is a tough challenge for chemists and biologists. 3 Carbohydrates are hydrophilic species and therefore difficult to be extracted from water by traditional pre-treatment methods. As they contain multiple hydroxyl groups, they are also hydromimetic, blending easily into a background of water molecules. 4,5 Synthetic receptors for specic carbohydrates recognition is a challenging yet highly impactful area of research. [6][7][8][9] Phenylboronic acid (PBA) and its derivatives, known be able to rapidly and reversibly interact with 1,2-or 1,3-diols in aqueous media, are the most viable candidates for carbohydrate receptor design. [10][11][12] However, the synthesis routes of receptors for carbohydrate sensors are usually complicated and tedious, and the efficiency and selectivity of the synthetic receptors, particularly ones that work in competitive solvents, remain a major challenge. The reasons for this are that the interactions of a receptor with the OH groups of a carbohydrate-derived substrate do not fundamentally differ from that with water molecules, which causes the cross-interference of the determination signals, and also the structural similarity of many carbohydrates, D-glucose and D-mannose, for example, differ in the conguration of only a single OH group on the ring. 5 In addition, the determination principles mainly depend on the physicochemical signal changes of the receptor exposed to the sample, such as uorescence, [13][14][15] swelling/ shrinking degree, [16][17][18] diffraction 19 and conductivity, 20 and so on. It inevitably means that: (1) the limits of detection of carbohydrate sensors mostly range from hundreds of micromoles to millimoles per litre. Given this, the potential for application in unconventional body uids containing low carbohydrate concentrations, such as interstitial uid extracted by iontophoresis, tears, saliva and urine and at intracellular concentrations at the single-cell level in metabolomic studies, [21][22][23][24] are infeasible. (2) The synthetic receptors are not capable of application for carbohydrate recognition in semisolid or solid biological tissues, which are the main components comprising organisms.
Carbon nanomaterials, such as carbon nanotubes (CNTs), have been explored extensively for carbohydrate-related biomolecule recognition in recent years. 25,26 Pristine carbon nanomaterials are characterized by low solubility, thus the surface properties of CNTs must be tuned not just to improve their water solubility but also to enable these versatile nanomaterials to interact selectively with biological systems in aqueous systems. Chen et al. recently reported a review, which highlighted the strategies for synthesis of functionalized carbon nanomaterials and their applications in biosensing and biomedicine. 27 As reported recently, functionalized CNTs could serve as excellent one-dimensional scaffolds for ligands, which can exhibit strong affinity towards lectin, 28 glucose 9 and glycan 29 in aqueous systems. In addition, a novel nanocomposite consisting of 3-aminophenylboronic acid and CNTs was synthesized and an impedance-cell sensor was constructed. 30 Herein, we fabricated an ultrasensitive SPME probe based on PBA functionalized CNTs, which enabled fast, quantitative and direct carbohydrate analysis in biouids or semi-solid biotissues, by coupling with gas chromatography-mass spectrometry (GC-MS). Firstly, PBA functionalized-CNTs were synthesized and utilized as carbohydrate nano-receptors. The hybrid, containing an appropriate ratio of nano-receptors to other biocompatible and acid resisting polyacrylonitrile (PAN) groups, was attached to a pretreated quartz ber through dipcoating method, forming a novel pH-controlled capture/release miniature probe for selective capture of carbohydrates. Owing to the 3D interconnected architecture formed by the stacking of PBA functionalized-CNTs in the coating, the proposed probe possessed excellent binding capacity toward carbohydrates (the enrichment factors were as high as 151). Simply by adjusting the pH of the eluent, this proposed probe was feasible to couple with GC-MS for carbohydrate separation and detection, which dexterously avoided the cross-interaction effect and simultaneously greatly improved the sensitivity for carbohydrate recognition (Scheme 1). Interestingly, the proposed probe was suitable to identify and differentiate carbohydrates in a multicarbohydrate system. Moreover, this carbohydrate probe was successfully applied to determinate glucose in bovine serum and human urine without any expensive enzymes or tedious pretreatment procedure. Importantly, the excellent biocompatibility and mechanical strength of the probe made it possible to directly immerse the probe into semi-solid biological tissues (plant leaf and stem) for in vivo carbohydrate recognition and continuous carbohydrate monitoring.

Results and discussion
Synthesis and characterization of the receptor Given the ultrahigh ratio of surface area to volume of carbon nanotubes (CNTs), and the extreme sensitivity of their surface atoms to any surface reaction events, functionalization of CNTs was used as the starting point. Herein, PBA functionalized-CNTs, with an external diameter about 40 nm ( Fig. 1A and S1 †), were synthesized and used as carbohydrate receptors. IR spectra (Fig. 1B) and XPS ( Fig. 1C and S2 †) conrmed the presence of PBA groups on CNTs. The affinity of the PBA functionalized-CNTs towards diol units was rst conrmed using adenosine as a test compound, which contains a pair of cis-diol groups and it has UV absorbance at about 260 nm. Deoxyadenosine was used as an interferent, which also has UV absorbance at about 260 nm while containing no cis-diol moiety. As shown in Fig. 1D, the PBA functionalized CNTs exhibited excellent selectivity toward adenosine, and the binding amount was depended on the exposure time (Fig. S3 †). In addition, the binding capacity toward adenosine was measured to be 50.9 AE 2.3 mmol g À1 . These results demonstrate that the synthesized receptor showed excellent selectivity toward cis-diol.  Binding capacity toward diol of the probe There are two probe design challenges: (1) to apply an appropriate auxiliary for xing the receptor on the solid substrate (quartz ber); (2) to prevent the auxiliary material from covering the receptor, otherwise, the covering auxiliary could block binding sites of the receptor. Herein, PAN (M w ¼ 150 000, dissolved in dimethylformamide 1 : 10, v/v), which is regarded as a biocompatible and acid resisting polymer, [31][32][33] was selected as the auxiliary for attaching the receptor to the pretreated quartz ber through a dip-coating method, while dimethylformamide was utilized as it evaporates at high temperature (120 C, 40 min) and so is suitable to create a porous structure of the coating ( Fig. 2A). In addition, as seen in Fig. 2A and B and S4, † the stacking of CNTs could form 3D interconnected pores compared with the nanoparticle stacking (we fabricated another PBA functionalized-carbon dots based probe as a nanoparticle stacking probe model, which was shown in Fig. 2C and D). This architecture possessed high specic surface area and facilitated mass transfer in the coating, which greatly enhanced the availability of PBA groups (Fig. 2E). Meanwhile, owing to the reversible binding of PBA groups-diol unit 10 and the high acid resisting ability of PAN, the bound diol unit was able to be released for further qualitative or quantitative analysis simply by adjusting the pH of the eluent (Fig. 2F). As shown in Fig. 2G, the extraction efficiency of the proposed probe toward diols was much superior to that of other probes widely used in biological analysis, including polydimethylsiloxane (PDMS) and C18. [34][35][36][37] In addition, the comparison result between the PBA functionalized probe and non-PBA functionalized probe indicated that the extraction performance of the proposed probe was substantially due to the PBA groups in the coating, which demonstrated that the PBA groups in the probe were available and provided a specic scaffold for carbohydrate binding.

Specicity of the probe toward carbohydrates
Selectivity is a critical parameter to evaluate the performance of the probe. We rstly studied the response of the proposed probe toward potential interfering substances coexisting in biouids, including various amino acids, aliphatic acids, glutathione and uric acid. As shown in Fig. 3, the extraction capacity of the probe toward these substances was negligible. The exclusive extraction of carbohydrate (glucose) resulted from the following reasons. Firstly, the carbohydrate possessed specic multiple hydroxyl groups structure while these coexisting substances have no such special structure. Secondly, as demonstrated in Fig. 1D and 2, the probe provided a specic scaffold for the diol unit, especially for the cis-diol. Thus, carbohydrates, which possess various 1,3 or 1,2 cis-diol units, could be easily captured by the scaffolds.

Glucose recognition in PBS
The performance of the prepared probe for carbohydrate assay was rstly evaluated in phosphate buffer solution (PBS). Glucose, a typical monosaccharide, was used as the carbohydrate model and 0.2 M acetic acid (pH z 2) was selected as the eluent. As shown in Fig. 4A, the binding reaction was completed within 20 min. Such a good performance was due to the 3D interconnected pores and xed orientation of the PBA molecules within the probe, which facilitated efficient mass transfer and complexing simultaneously.
Under the optimized conditions, the probe was applied for glucose determination in PBS solution. Due to the enrichment effect of the probe (see below) and the high resolution detector, the linear range of the probe was found to range from 1 to 100 mM for glucose determination (Fig. 4B), with a limit of detection of 0.12 mM (signal-to-noise ratio of 3). To our knowledge, the linear range and the detection limit of the proposed probe were much better than of most previous boronic acid based sensors (Table S1 †). A higher sensitivity for glucose assay is important not only in low concentration biouids, such as tears, saliva and urine, but also in high glucose level biouids, such as blood (several to tens of millimoles per liter). The reason is that highly sensitive probes would allow a sufficient sample dilution during assay, which can effectively reduce interference from the complicated matrix (blood/serum). 38 It is noteworthy that the whole assay procedure, consisting of binding, elution and detection, required less than 2 h. In addition, the reusability of the probe was evaluated. It was shown that the extraction efficiency did not signicantly change even aer 20 times reuse (Fig. S5 †).

Multi-carbohydrate recognition in PBS
Carbohydrates are one of the most abundant molecules that comprise human life and many kinds of carbohydrate are present. The approaches based on sensors for carbohydrate recognition have some drawbacks, one is that it is not capable of qualitative or quantitative recognition of multi-carbohydrate in biouids. In parallel with the emergence of glycomics, novel assays for fast and accurate carbohydrate recognition in multicarbohydrate biouids is required. [39][40][41][42] Here, owing to excellent separation ability of chromatography as well as the high Fig. 3 The response of the probe toward various potential interfering substances. The mixture, which contained glucose, various amino acids, aliphatic acids, glutathione and uric acid (concentrations ¼ 10 mM), was extracted by the probe for 40 min. The result showed that the probe presented specific selectivity towards carbohydrate (glucose). The inset shows the response of the probe toward glucose in the solution without and with interfering substances. resolution of mass spectrometry, the proposed probe coupled with GC-MS was proved to be feasible to recognize different carbohydrates in a multi-carbohydrate PBS solution, with linear ranges from 0.5 to 20 mM (Fig. 5A), and much superior to the low qualitative or quantitative ability of the previous boronic acid based sensors.
Owing to the unique 3D interconnected architecture in the coating, the enrichment factors, dened as the ratio of the carbohydrate concentrations in probe and in matrix, were measured to range from 63 to 151 (Fig. 5B). Generally, the probe showed higher enrichment capacity toward hexoses than pentoses. The reasons we propose are: (1) PBA can bind with cis-1,2-or 1,3-diols to form a diol-phenylboronate complex with either veor six-membered ring systems, respectively, 43,44 (2) the binding amount is largely dependent on the probability of directional collision. From this point, hexoses, which have an additional hydroxy unit compared with pentoses, should more readily complex with PBA. It was noteworthy that no selectivity against structural similar carbohydrates, such as mannose, glucose and galactose, was required in this approach since the probe was easy to couple with GC-MS.

Assay in serum and urine
Given the simplicity and ultra-sensitivity of carbohydrate determination with the proposed probe, the approach can be viewed as being ideal for monitoring carbohydrates in real biouids. As a proof of concept, bovine serum was rstly used as a biouid model. Considering the high glucose level in serum, 15 mL bovine serum was diluted 100 times with the PBS solution, and then was transferred into a 2 mL vial. The probe was directly immersed into the serum for glucose determination without any expensive enzymes or tedious pretreatment procedure. As shown in Table 1, the glucose level obtained by the proposed probe was in good agreement with the values measured by a commercial blood glucose monitor. Beneting from the ultrasensitive property of the proposed approach, the probe was also successfully applied for glucose determination in urine, typically a trace glucose containing sample (Table 1).

In vivo carbohydrate recognition
Previous fully-implantable sensors, embedded in the body for carbohydrate recognition, are seriously challenged mainly due to the insufficient physicochemical signal intensity required for transdermal detection. 45 Fortunately, the proposed probe could overcome this tough challenge due to the different detection principle. Thus, besides the above in vitro analysis, the other major novel application of the proposed probe was in vivo carbohydrate recognition in semi-solid biotissues, such as plant stem and leaf. Fig. 6A briey presents the in vivo sampling procedure in plant tissues. To achieve this goal, the probe should be capable of resisting the adhesion of biological macromolecules on the surfaces. Otherwise, the adhered biological macromolecules would block the binding sites of the probe and alter the ionization efficiencies when the macromolecules were desorbed in the desorption solvents. It was demonstrated through MALDI-TOF MS that no macromolecules were present in the eluent of probe exposed in aloe leaf or Malabar spinach stem for 30 min (Fig. 6B and S6 †). Moreover, the excellent mechanical strength of the probe, which could be observed in ESI movie 1, † make it suitable for direct immersion into semi-solid biotissues. Regarding the carbohydrates detected in this study, glucose was the main monosaccharide detected in Malabar spinach while rhamnose, mannose, glucose and galactose were detected in aloe without any plant sacrice (Fig. 6C). The abundant monosaccharide species detected in aloe leaf with the proposed assay was in good agreement with the previous analysis aer several tedious pretreatments, 46,47 which demonstrated the feasibility for in vivo  The average detected concentration of glucose in serum was 5.7 mM with the proposed probe, which was in good agreement with the values measured by a commercial blood glucose monitor (5.9 mM).
Notably, glucose at a very low level (16.6 mM) was detectable using the proposed probe.
analysis. Finally, the probe was used for noninvasive and longterm in vivo continuous carbohydrate monitoring in aloe leaf (Fig. 6D).

Conclusions
In summary, a novel SPME probe based on PBA functionalized-CNTs is proposed for fast and ultrasensitive determination of carbohydrates in biouids and semi-solid biotissues. The proposed approach was demonstrated to be much superior to previous carbohydrate sensors. It exhibited several signicant advantages, including higher sensitivity, wider linear range, and excellent qualitative ability. Unlike the method based on carbohydrate sensors, no complicated and tedious synthesis route of the receptor was needed in the proposed approach. Moreover, the probe was capable of direct in vitro or in vivo determination of multi-carbohydrates in complex real sample matrices, and the total analysis procedure was time-efficient (less than 2 h). Notably, the preparation approach of the probe can be applicable to other nanomaterials. In conclusion, this approach opens up new avenues for facile and efficient tracking and recognition of carbohydrates in bio-samples and could be a promising approach for important applications such as glycomics.  purchased from Sigma Aldrich (Shanghai, China) and human urine was obtained from Centre for Disease Prevention and Control of Guangdong Province (Guangzhou, China). All the plants used in this study were cultivated from seeds in the plant growth chamber (Conviron A1000, Canada).

Synthesis of the PBA-functionalized CNTs
Firstly, the raw sample of CNTs was reuxed in nitric acid for 11 h, and the carboxylated CNTs were ltered and washed with deionized water until pH 7, and dried in vacuum oven. Then 300 mg of carboxylated CNTs were stirred in 60 mL of a 20 : 1 mixture of SOCl 2 and DMF at 70 C for 24 h. Aer the acyl chlorination, the CNTs were centrifuged and washed with anhydrous THF six times, followed by drying under vacuum. Then the acyl-chlorinated CNTs were reacted with 100 mL diamine solution at 100 C for 48 h. Aer cooling to room temperature, the amino functionalized-CNTs (A-CNTs) were obtained and washed with ethanol ve times to remove excess diamine. Lastly, 100 mg A-CNTs, 0.4 mmol 4-CPBA, 0.5 mmol DIC, 0.5 mmol DIEA and 0.5 mmol NHS were dissolved in a mixed solution of dichloromethane and DMF. The reaction solution was kept stirring for 24 h at room temperature. Then, diethyl ether was added to terminate the reaction followed by ltration and washing with diethyl ether, water and methanol. The product was dried under vacuum to obtain the PBA functionalized-CNTs.

The selectivity of PBA-functionalized CNTs
For demonstration the selectivity of the PBA-CNTs toward cis-1,2-diol compounds, adenosine and deoxyadenosine were used as model compounds. 2 mg PBA functionalized-CNTs was added to 1 mL solution of 1 mg mL À1 adenosine or deoxyadenosine. The tubes were shaken on a rotator (400 rpm) for 1 h at room temperature. Then the suspension was centrifuged and the collected PBA functionalized-CNTs was rinsed with 1.5 mL of the PBS solution (pH 8.5) for 6 times each. Aerwards, the PBA functionalized-CNTs was resuspended in 1 mL of 0.2 M acetic acid solution and eluted for 1 h on a rotator with 600 rpm speed. Finally, the PBA functionalized-CNTs was recollected by centrifugation and the eluates were collected by pipetting carefully. The eluates were used for UV analysis.

The binding capacity of the receptor
For measurement of the binding capacity, a series of adenosine solutions (1.00, 0.50, 0.10, 0.050, 0.0010, 0.00050 mg mL À1 ) was prepared. The absorbance of the six solutions was detected to draw the standard curve. 2 mg PBA functionalized-CNTs were added to 1.5 mL of 1.0 mg mL À1 adenosine solution in a 2 mL plastic tube. The solution was shaken on a rotator for 12 h at room temperature to ensure the binding sites were entirely complexed with adenosine. The following centrifugation and elution procedures were as above. According to the measured absorbance of the eluent and the standard curve, the concentration of the eluate was obtained, from which the binding capacity was calculated. The binding capacity of PBA functionalized-CNTs was measured to be 50.9 AE 2.3 mmol g À1 .

Preparation of the probe based on PBA functionalized-CNTs
Quartz bers (QFs) were cut into 4-5 cm segments followed by sonication in water, menthol and acetone. Aer sonication, the QFs were then soaked in 0.1 M sodium hydroxide for 30 min to activate the surface, and the excess sodium hydroxide was then neutralized with hydrochloric acid. Finally, the OFs were dried at room temperature. 100 mg PAN was fully dissolved with 1 g anhydrous DMF in a 1.5 mL plastic tube through 1 h sonication. 40 mg PBA functionalized-CNTs was then added to the plastic tube. Another 30 min sonication was conducted to form the dispersive slurry. The pretreated QFs were dipped into the slurry and removing them slowly, a uniform coating of slurry of PAN and PBA functionalized-CNTs with 1.5 cm length was prepared on the surface of the QFs. The QFs were dried under owing nitrogen, and nally cured for 40 min at 120 C, which facilitated DMF to evaporate and ensure better adherence of the coating to the QFs.

Binding capacity of the probe
To study the advantage of the nanotube stacking of the coating, another probe based on PBA functionalized-carbon dots, which possessed nanoparticle stacking ( Fig. 2C and D), was used as a reference. The synthesized method of PBA functionalized-carbon dots was referenced to Shen and Xia. 48 Briey, 0.2 g of phenylboronic acid was dissolved in 20 mL of ultrapure water, followed by adjusting the pH to 9.0 by adding 0.1 M NaOH under stirring, then bubbling nitrogen gas for 1 h to remove dissolved O 2 . Finally, the solution was transferred to a Teon-lined autoclave chamber and heated to 160 C for 8 h. TEM images and uorescence spectra of the PBA-carbon dots are provided in Fig. S7. † The preparation of the probe based on PBA functionalized-carbon dots, including material dosage and preparation process, was consistent with the method mentioned in the previous section. The characterizations of micro-morphologies were shown in Fig. 2C and D. For evaluation of the binding capacity, each kind of probe was immersed into a glucose aqueous solution (10 mM, 1.5 mL) and then shaken on a rotator. Aer 60 min, the probe was removed and rinsed with deionized water for 30 s followed by drying with a Kimwipe tissue. Finally, the bound glucose on the probe was then eluted in 1.5 mL 0.2 M acetic acid solution, and the concentration of glucose was detected by GC-MS.
Comparison of extraction performance with other commonly used biological probes PDMS and C18 commercial probes (both 45 mm in thickness) were obtained from Supelco Inc (Shanghai, China), and a further CNTs probe without PBA modication was prepared through the same method mentioned above in our lab. The concentration of glucose used was 50 mM. All the experimental parameters and relative operating process were the same as for the PBA modied CNTs probe measurements.

Evaluation of the performance in PBS solution
For glucose assay in PBS solution, a series of glucose solutions (dissolved in PBS, 1.0, 5.0, 10.0, 20.0, 50.0, 100.0 mM) was prepared in a 2 mL brown vial with cap gasket (polytetra-uoroethylene). The procedure of introduction and xation of the probe in PBS solution was as follows: a steel needle of a hypodermic syringe head was pierced into the cap gasket to create a hold. The end of the QFs, which is the opposite end of the coating, was then inserted into the cap gasket. Aerwards, the cap with QFs was screwed (Fig. S8 †). The extraction was performed on a rotator with 400 rpm speed for 20 min. Aer extraction, the cap was removed and the probe was then rinsed with deionized water for 30 s and dried with a Kimwipe tissue. Subsequently, the probe was immersed into the glass vial (250 mL 0.2 M acetic acid) and eluted for 30 min (optimized in Fig. S9 †) on a rotator with 600 rpm speed. The eluent was processed with a simple derivatization prior to introduction into GC-MS for glucose determination. For multi-carbohydrate assay in PBS solution, a series of carbohydrate solutions, containing ribose, rhamnose, mannose, glucose and galactose (dissolved in PBS, 0.50, 2.5, 5.0, 10.0, 20.0 mM), was prepared. The assay procedure was the same as for the glucose solutions.

Ex vivo glucose assay in bovine serum and human urine
The bovine and human urine was stored at À80 C prior to analysis. For assay in bovine serum sample, 15 mL thawed bovine serum was diluted to 1.5 mL PBS solution in a 2 mL brown vial. For assay in human urine sample, due to the trace glucose contained in urine, 1.5 mL thawed human urine was directly transferred into 2 mL brown vial by pipetting without further dilution. The probe was then directly immersed into the serum or urine sample for glucose assay without any expensive enzymes and tedious pretreatment procedure, and the assay procedure was the same as that in the previous section.

In vivo carbohydrate assay in plants
The customized hollow steel needle was stabbed into the leaf or stem of the plant to a depth of about 2 cm. The probe was then inserted into the needle and reached to the end of the needle. Subsequently, the needle was carefully withdrawn back to let the probe be exposed in the plant tissues. Aer a certain duration, the needle was put back into the plant under the guidance of the probe to a depth of about 1.5 cm. Then, the probe was withdrawn from the needle, and the needle was removed. The total sampling duration was controlled to be 30 min. The probe were then rinsed with deionized water three times (60 s for each time) and dried with a Kimwipe tissue. Subsequently, the probe was immersed into a glass vial (250 mL 0.2 M acetic acid) and eluted for 30 min on a rotator with 600 rpm speed. The eluent was processed with derivatization and then introduced into GC-MS for carbohydrate determination.

GC-MS analysis
The eluent for carbohydrate analysis needs a simple derivatization prior to GC-MS analysis. Briey, 2% 0.2 mL sodium borohydride solution (dissolved in aqueous ammonia) was added into the eluent. Aer reaction for 20 min at 40 C, 0.4 mL acetic acid, 0.3 mL methylimidazole and 1 mL acetic anhydride was added to the solution followed by another 10 min reaction.
Finally, the carbohydrate derivatives were dissolved in 500 mL dichloromethane. The carbohydrate detection was performed on an Agilent 6890N gas chromatograph equipped with a MSD 5975 mass spectrometer (GC-MS) and electron-impact ionization (EI). A split/splitless-type injector was used for sample introduction. Chromatographic separation was carried out with a HP-5MS capillary column (30 m Â 250 mm Â 0.25 mm, Agilent Technology, CA, USA). The inlet temperature was 240 C, and the oven temperature programs were as follows: the initial oven temperature was 140 C (held for 0.5 min), ramped at 30 C min À1 up to 190 C (held for 5 min), and ramped at 2 C min À1 up to 210 C (held for 2 min). Helium was used as the carrier gas at a constant ow rate of 1.2 mL min À1 . The MSD was operated in the electron impact ion (EI) mode with a source temperature of 230 C. The electron energy was 70 eV and the lament current was 200 A.

Biochemical analyzer for glucose assay
To evaluate the feasibility of the proposed probe for determination of glucose, the concentrations of glucose in human serum were assayed by an enzymatic (hexokinase) test using a PUZS-300 automatic biochemical analyzer as referenced by Shen and Xia. 48 Briey, the sample was rstly added into the sample cup, which was then placed in the sample frame for measurement aer setting the parameters. Under the catalytic effects of hexokinase, glucose and adenosine triphosphate (ATP) can react and form glucose-6-phosphate and adenosine diphosphate (ADP). The former can dehydrogenize and form 6phosphate glucose acid in the presence of glucose-6 phosphate dehydrogenase. At the same time, nicotinamide adenine dinucleotide phosphate (NADP) is reduced and forms nicotinamide adenine dinucleotide phosphate (NADPH). The production rate of NADPH is proportional to the concentration of glucose, which can be monitored by the absorbance at 340 nm and so allow measurement of the glucose concentration.