Nano Pt@ZIF8 Modiﬁed Electrode and Its Application to Detect Sarcosine

Sarcosine is a newly discovered effective biomarker for prostate cancer. However, the low concentration of sarcosine in tissue cells, plasma or urine blocks the development of sarcosine biosensors. In this manuscript, porous zeolitic imidazolate framework-8 (ZIF8) was synthesized and was used as carriers to load nano platinum (Pt@ZIF8). The porous structure of ZIF8 helped to stabilize the nano platinum and keep its high catalytic activity. The Pt@ZIF8 modiﬁed sarcosine biosensor had good response toward sarcosine due to its unique structure and morphology. The linear range of the as prepared biosensor is from 5 to 30 μ M, which is consistent with the detection demand. The prepared sarcosine biosensor is suitable to be developed as a kind of portable diagnostic facilities for prostate cancer.

Prostate cancer (PCa) is one of the major diseases that influence the health and life expectancy of men over 50 years' old. 1 However, the prognosis is excellent if early prostate cancer (EPCa) is caught early. The 5-year survival rate for EPCa is almost 100%. So an obvious question arises: can we identify PCa early enough? Yes, the clinical method for screening EPCa is prostate specific antigen (PSA) test. 2 But unfortunately, PSA test has been found to be unreliable in practice. Both the false negative rate (15.2%) and false positive rate (30%) are very high, though it is the most widely used clinical method for EPCa. 3 Human beings have to look for better biomarkers for prostate cancer. 4 In 2009, scientists found that sarcosine in tissue cells, plasma or urine is an effective biomarker for prostate cancer. 4 By measuring the concentration of sarcosine in urine or blood plasma or tissue cells, it is easy to distinguish healthy individuals, benign prostate disease, clinically localized prostate cancer and metastatic prostate cancer. Furthermore, sarcosine can be detected non-invasively in urine. This report immediately caused a worldwide research upsurge. More and more results show that sarcosine in urine is a more effective biomarker for prostate cancer. [5][6][7][8] Although sarcosine has been recognized as an effective biomarker for PCa, it is still very hard to be detected due to its very low concentration and complicated composition. 4 The concentration of sarconsine in urine ranges from several micromolar per liter to several dozen micromolar per liter. 4 With such a low concentration, the analytical methods available for direct determination of sarcosine in urine often include chromatography and tandem mass spectrometry. 9 The major disadvantages of these methods include high instrumentation costs, complicated sample preparation and experienced operator requirements. So they are difficult to be applied as a wide range of routine analysis. 10,11 Due to the high cure rate of early PCa, the accurate measurement methods for trace amount of sarcosine with properties of simple, fast and low cost is especially important. Some efforts have been made toward the low-cost bioanalytical tools for sarcosine diagnostics. 1,10,12 On the other hand, amperometric biosensor has the advantages of simple, rapid and low cost, and has played an important role in disease diagnosis. [13][14][15][16] The most widely used amperometric biosensor is portable blood glucose meter, which has greatly facilitated the patients and medical staff. However, the study of amperometric sarcosine biosensor is very limited. 1,[17][18][19] Here we report a sarcosine biosensor with excellent performance. Zeolitic imidazolate framework-8 (ZIF8), a kind of metal organic framework (MOF) materials, was synthesized successfully and was used as carriers to load nano platinum (Pt@ZIF8). The porous structure of ZIF8 helped to stabilize the nano z E-mail: gezc@szu.edu.cn platinum and keep its high catalytic activity, and lead to high sensitivity of the prepared sarcosine biosensor.

Experimental
Chemicals and reagents.-Sarcosine oxidase (SOx) was purchased from J&K scientific company, the activity was 37 units mg −1 . Sarcosine was purchased from Acros organic company. Poly Instrumentation and measurements.-The scanning electron microscopy (SEM) photographs were measured on a Hitachi SU-70 electron microscopy, the accelerating voltage of electron beam was 5 kV. Mapping analysis was performed by energy dispersive spectrometer (EDS) with 15 kV accelerating voltage. Samples for SEM and EDS measurements were prepared by adhering a tiny bit of the powder on carbon conductive tape. The crystal structure of the obtained microstructure were verified by X-Ray diffraction (XRD, D8 ADVANCE). Cyclic voltammetry (CV) and amperometric measurements were carry out by using a CHI 660E electrochemical workstation (Shanghai Chen Hua Instrument Co. Ltd). All experiments were proceeded in a three electrode cell. The counter electrode was platinum electrode and the reference electrode was saturated calomel electrode (SCE). All experiments were conducted at room temperature (about 298 K).
The synthesis of ZIF8 and Pt@ZIF8.-The synthetic method of the ZIF8 is according to the steps reported by Venna et al. 20 Briefly, a methyl alcohol solution of 2-methylimidazole (3.3g, 70ml) was added into a methyl alcohol solution of Zinc nitrate hexahydrate (1.5g, 70ml), and the mixture was stirred at room temperature for 24 hours to obtain a milky solution. The milky solution was centrifugalized to obtain powder samples of ZIF8. The pristine ZIF8 powders was washed 3 times with 50 mL of methanol, and was dried at 353 K for 12 h.
Pt nanoparticles which loaded on the ZIF8 (Pt@ZIF8) were synthesized by using the method of in situ reduction. Firstly, 0.1 g ZIF8 was added into the ethanol solution of H 2 PtCl · 6H 2 O (0.2g, 20ml) and was stirred for 24 h. Then an ethanol solution of sodium borohydride was added into the solution with stirring for 60 min. The obtained black suspension (Pt@ZIF8) was centrifuged for 10 min at 10000 rpm (room temperature) and was washed 3 times with ethanol. The as prepared Pt@ZIF8 powder was heated at 353 K for 12 h under Ar atmosphere.

Results and Discussion
The surface morphology and structure of ZIF8 and Pt@ZIF8.-The morphology of the as prepared ZIF8 and Pt@ZIF8 were taken by a Hitachi SU-70 electron microscopy and were shown in Fig. 1. The acceleration voltage was 5 kV and the magnification is 100,000. Fig. 1a shows that the morphology of the as prepared ZIF8, and Fig. 1b shows the morphology of the as prepared Pt@ZIF8. Both ZIF8 and Pt@ZIF8 are uniform and integrated nanoparticles. The average particle size is 33 nm for ZIF8 and 54 nm for Pt@ZIF8. So we can deduce that there are many reduced Pt nanoparticles uniformly loading on the surface of the ZIF8, and the average diameter of the Pt nanoparticles are estimated to be 10 nm. On the other hand, compared Fig. 1a with Fig. 1b, the number of voids on Pt@ZIF8 is much less than in ZIF8. This also shows that the reduced Pt nanoparticles uniformly loading on the surface of the ZIF8. The elemental analysis of the Pt@ZIF8 was made by the energy dispersive spectrometer (EDS) and showed in Fig. 1c 2 presents the X-ray powder diffractometer (XRD) patterns of the prepared ZIF8 and Pt@ZIF8. The XRD pattern of the as prepared ZIF8 is consistent with the Reference 20. The XRD pattern of the as prepared Pt@ZIF8 consistent with Pt, and also consistent with ZIF8. The XRD patterns verify the successful preparation of ZIF8 and Pt@ZIF8.
Electrochemical characterization of Pt@ZIF8 modified electrode.-In cyclic voltammetry (CV) measurements, the potential of oxidation and reduction peak in reversible process was an important factor to characterize the electrochemical properties of the electrode. To characterize the electrochemical property of the Pt@ZIF8 modified electrode, CV measurements were done in Fe(CN) 6 4−/3− solution. In this work, the CV curves of the bare glassy carbon electrode and modified electrodes were done in 5 mM of potassium ferricyanide solution (adding 0.01646 g K 3 [Fe(CN) 6 ] to 10 ml phosphate buffer solution (PBS) with a pH value of 7.0), and were presented in Fig. 3. The glassy carbon electrode was polished with Al 2 O 3 powders and ultrasonicated in alcohol and water solution before use. The CV curve of the bare glassy carbon electrode in PBS solution shows only the non-Faraday current. In Fe(CN) 6 4−/3− solution, the oxidation peak of the bare glassy carbon electrode caused by the Fe 3+ /Fe 2+ redox couples occurs at 0.25 V (vs. SCE) and the reduction peak appears at 0.11V (vs. SCE), the E is 0.14V. However, the oxidation peak of the Pt@ZIF8 modified electrode caused by the Fe 3+ /Fe 2+ redox couples occurs at 0.25 V (vs. SCE) and the reduction peak appears at 0.14 V (vs. SCE), the E is 0.11V. The E reduced from 0.14 V to 0.11 V. Though the E of ZIF8 modified electrode is smaller than the Pt@ZIF8 modified electrode, it has smaller peak current. The peak current of Pt@ZIF8 modified electrode is much larger than the bare glassy carbon electrode and ZIF8 modified electrode, this is mainly caused by the large surface area of Pt nanoparticles dispersed on the modifying layer. According to Randles-Sevcik equation 21 I p = 2.69 * 10 5 AD 1/2 n 3/2 ν 1/2 c where A is the electroactive area of the electrode (cm 2 ); D is the diffusion coefficient of the molecules in solution, and is 6.70 ± 0.02 × 10 −6 cm 2 s −1 ; n is the number of electrons participating in the reaction, and usually is equal to 1; c is the concentration of the probe molecule in the solution (5.0 mM), and ν is the scan rate of the CV measurement (0.1 V s −1 ). Thus the electroactive area of the Pt@ZIF8 modified electrode is estimated as 35.8 mm 2 , which is much higher than the electroactive area of the ZIF8 modified electrode (26.7 mm 2 ) and the bare glassy carbon electrode (22.3 mm 2 ). This showed that the Pt@ZIF8 nanoparticles has excellent electrocatalytic activity.
Amperometric detection of sarcosine with Pt@ZIF8 modified bisosensor.-The Pt@ZIF8 modified biosensor was fabricated by casting a layer of Pt@ZIF8 over the surfaces of glassy carbon electrode and immobilizing sarcosine oxidase (SOx) on the electrode with glutaraldehyde solution (0.5%) and Nafion solution (0.5%). All chronoamperometric experiments were proceed in 10.0 ml of PBS solutions with stirring.
At an applied potential of 0.4 V, with the successive injections of 5 or 10 μM sarcosine into PBS solution, the oxidation current increased greatly and rapidly reached to a steady state in very short time. The well-defined curve displays its high sensitivity and gradient. The resulting calibration plot for sarcosine over the concentration range from 5 μM to 50 μM is presented in the inset of Fig. 4. It shows that the as prepared biosensor could work linearly from 5 to 30 μM. The corresponding regression equation of the linear plot is: i/nA = 5.36 + 4.05 c/μM, R = 0.996. The sensitivity is thus estimated as 4.05 nA μM −1 . The detection limit is estimated to be 1.06 μM (S/N = 3) according to the calibration curve.
The linear range, limit of detection (LOD) and applied potential (V vs. SCE) in some typical amperometric sarcosine biosensors are shown in Table I with those mentioned sarcosine biosensors. On the other hand, the concentration of sarcosine in human urine ranges from several micromolar per liter to several dozen micromolar per liter. Very few of the reported results is consisted with this range. More efforts are needed to provide better amperometric sarcosine biosensors. Possible interferents such as ascorbic acid, uric acid, L-cysteine, glycine, fructose, maltose, lactic acid, citric acid, sucrose, calcium chloride are added into 50 μM sarcosine solution to measure the response signal. All other possible interferents do not substantially change the response signal except the ascorbic acid and L-cysteine. In 50 μM of sarcosine solution, the response signals of 0.1 mM ascorbic acid and 0.1 mM L-cysteine are comparable to the signal from sarcosine. This is a question for low concentration substrate test in real uric or serum samples. We will pay close attention to this question in the future studies.
The longtime activities of the Pt@ZIF8 modified sarcosine biosensor were tested in two weeks. It shows that the sarcosine biosensor could work in 3 days (only lost 15% of its initial activities). But in two weeks, it lost almost 50% of its initial activities. Compared with the long lifetime of glucose biosensor (almost 2 months), its believed that the immobilized SOx has very low stability. Great efforts should be done to prolong the lifetime of sarcosine biosensor in the future studies.

Conclusions
This work presents an amperometric sarcosine biosensor consisting of nano Pt@ZIF8 and sarcosine oxidase. Due to the unique 3D network structure of ZIF8, homogeneous platinum nanoparticles are obtained and the high electrocatalytic activity of platinum nanoparticles is maintained. The Pt@ZIF8 nanoparticles modified biosensor exhibits excellent sensitivity and could detect sarcosine in the concentration scope of micro molar. The linear range of the sensor is from 5-30 μM, which is consisted with the concentration range of sarcosine in human blood plasma or urine (from several micromolar per liter to several dozen micromolar per liter). This novel nanostructure provides excellent electrode materials for the development of high performance biosensors and other bioelectrochemical devices.