Detection of Phenylketonuria Markers Using a ZIF-67 Encapsulated

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Biological and Medical Applications of Materials and Interfaces

Detection of PKU Markers Using a ZIF-67 Encapsulated PtPd Alloy Nanoparticles (PtPd@ZIF-67) Based Disposable Electrochemical Microsensor Xinyue Xu, Dongqing Ji, Yuan Zhang, Xinghua Gao, Pengcheng Xu, Pengfei Hu, Xinxin Li, Chung Chiun Liu, and Weijia Wen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b05431 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 17, 2019

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ACS Applied Materials & Interfaces

Detection of PKU Markers Using a ZIF-67 Encapsulated PtPd Alloy Nanoparticles (PtPd@ZIF67) Based Disposable Electrochemical Microsensor Xinyue Xu,†,⊥ Dongqing Ji,†,⊥ Yuan Zhang,*,† Xinghua Gao,† Pengcheng Xu,‡ Xinxin Li,‡ Chung-Chiun Liu§ and Weijia Wen† †Materials

Genome Institute, Shanghai University, Shanghai 200444, China. E-mail:

[email protected] ‡State

Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and

Information Technology, Chinese Academy of Sciences, Shanghai 200050, China §Department

of Chemical Engineering, Case Western Reserve University, Cleveland, OH 44106,

USA

ABSTRACT Phenylketonuria (PKU) is a common disease in congenital disorder of amino acids metabolism, which can lead to intellectual disability, seizures, behavioral problems, and mental disorders. We report herein a facile method to screening for PKU by the measurements of its metabolites (markers). In this work, an disposable electrochemical microsensor modified with

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ZIFs (zeolitic imidazole frameworks) based nanocomposite is constructed, in which ZIF-67 crystals are encapsulated with PtPd alloy nanoparticles (NPs) forming the nanocomposite (PtPd@ZIF-67). According to electrochemical measurements, the PtPd@ZIF-67 modified microsensor shows good responses and selectivity to phenylpyruvic acid and phenylacetic acid, while almost no response towards other amino acid analogues is observed. Here, a new sensing mechanism based on acylation reaction between imidazole linker in ZIF-67 and carboxyl in PKU markers has been proposed and verified through FT-IR study. Moreover, the encapsulated PtPd NPs elevate electron transfer capability of PtPd@ZIF-67 modified microsensor and further improve the electrochemical sensing performance. Finally, we demonstrate that the developed PtPd@ZIF-67 modified microsensor has the possibility to sensing of PKU markers with high response and good specificity, and may be extended to exploit the point-of-care (POC) rapid PKU screening.

KEYWORDS ZIF-67, encapsulation, metal nanoparticles, electrochemical sensing, PKU markers, disposable microsensor

INTRODUCTION Phenylketonuria (PKU) is the most common disease in congenital disorder of amino acids metabolism, and often found in children and adolescents.1 PKU can harm the neonatal nervous system, so untreated children often exhibit severe mental retardation as well as physical and behavioral abnormalities.2 More than that, the later stages of children with PKU will cause epileptic seizures and eventually lead to death.3 Although PKU is not curable, the early recognition and effective treatment of children with PKU could prevent irreparable damage to


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the nervous system, thus relieving the patient's distress.4 Most patients are diagnosed via newborn screening, and the non-invasive blood or urine sampling is more appropriate due to their convenience and harmless for newborns.5 Moreover, the reliable point-of-care (POC) testing of the relevant metabolites (markers) may expedite the diagnosis and management of the underlying medical conditions. The detection of PKU markers in body fluid have been done with bacterial inhibition assay,6 immunoassays using fluorescence,7 electrochemical methods under the assistance of aptamer or DNA,8,9 as well as amino acids measurement using high performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS).10 The earliest use of phenylalanine to screen newborns is the bacterial inhibition method. Guthrie proposed to identify phenylketonuria by using Bacillus subtilis due to its growth is associated with phenylalanine.11 However, this method usually needs a long time for sample processing, and the presence of antibiotics in the blood of children with PKU would inhibit the growth of Bacillus subtilis. Among the other developed methods, electrochemical analysis with the advantages of rapid response, sensitivity and simple to handle has the potential to be used for the development of next generation of clinical diagnostic technology.12 Since PKU is a disease caused by deficiency of the metabolic enzyme phenylalanine hydroxylase (PAH), most of reported electrochemical assays are based on the measurements of PAH.13,14 In this way, complicated handling procedures and the requirement for special care with enzymatic activity make these methods difficult for continuous in situ analysis and less robust. Accordingly, the direct detection of PKU markers without the employment of bio-recognizers would avoid these limitations and be more suitable for trace sample analysis.


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To achieve this, a sensing electrode layer with high sensitivity and good specificity could be an ideal substitute for biological molecular recognition system. One such new materials class is known as zeolite imidazolate framework (ZIFs) which is composed of imidazolate-based linkers and transition metal atoms (mostly, Zn (II) or Co (II)).15 ZIFs are a sub-family of metal organic frameworks (MOFs), and have the similar geometrical features to those of zeolites.16 Thus, ZIFs generally display properties that combine the advantages of both zeolites and MOFs, such as well-defined pore structure, large surface area and structural flexibility.17 In addition, the imidazolate linkers, especially the imidazole nitrogen site within the porous frameworks endows ZIFs excellent selective adsorption properties and catalytic activities, making them good candidates for chemical sensing applications.18 Beyond that, the porous ZIFs enable the encapsulation of functional species into their cavities forming new types of composite materials, and also allow chemical species free access to the embedded materials that display enhanced properties.19-22 After encapsulation of nanosized guests, such as metal nanoparticles (NPs), quantum dots (QDs), polymers and biomacromolecules in ZIFs could lead to the development of new functional materials for gas storage & separation,23,24 drug delivery,25 catalysis,26 energy storage,27 and sensors.28 Herein, an electrochemical microsensor system has been processed by screen printing technology and used as sensing platform. Then a nanocomposite constituted with ZIF-67 encapsulated PtPd alloy NPs (PtPd@ZIF-67) is locally deposited onto working electrode of the fabricated microsensor, and employed as the sensing layer. For the sensor measurements, four PKU markers including phenylalanine, phenylpyruvic acid, phenylacetic acid and phenyllactic acid are implemented the experiments, so that the results obtained from multiple markers could be mutually corroborated and provide a good accuracy.


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EXPERIMENTAL Synthesis of surface modified PtPd alloy NPs. Firstly, 7 mL of ethylene glycol (EG) solution containing 30 mM polyvinylpyrrolidone (PVP, MW=24000) was added in a 25 mL flask and heated up to 160 °C under stirring and refluxing. 1 mL of EG solution containing 80 mM Na2PdCl4 and another 1 mL of EG solution containing 80 mM K2PtCl4 were simultaneously and rapidly added into above solution. The reaction could be quenched as soon as the color of solution was changed to black, and the black precipitate was collected by centrifugation. For the following encapsulation process, it is mainly based on the surface modified PVP molecules. The PVP-modified PtPd NPs were collected by washing with methanol for one time, and then dispersed in 150 mL methanol. The concentration of PtPd NPs in the solution is about 0.07 g/L. The encapsulation procedure for PtPd@ZIF-67 nanocomposite. Typically, 2 mL of the above PVP-modified PtPd NPs solution, 6 mL of 2-Methylimidazole methanol solution (0.8 M) and 20 mL of CoCl2·6H2O methanol solution (1 mM) were mixed together. The resultant solution was incubated at 50 °C for 20 hours to allow the heterogeneous nucleation and growth of ZIF-67. After the reaction is completed, the precipitate was collected by centrifugation and washing with methanol for several times. The final PtPd@ZIF-67 nanocomposite was obtained after drying under vacuum at 60 °C overnight. The content of PtPd NPs in nanocomposite is about 13.7%, which is calculated based on the concentration of prepared PtPd NPs solution and the weight of obtained nanocomposite. Sample Characterization. Transmission electron microscopy (TEM) characterization was performed with a FEI Tecnai G20 microscope operated at 200 kV. Scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray spectrometry (EDXS) elemental maps were determined by a JEM 2100F microscope. The phase and crystalline


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structure of sample were measured through X-ray diffractometer (XRD, Bruker, D2 PHASER) with a Cu Kα radiation (λ=1.5418 Å) as X-ray source. Diffraction data were collected at the scan rate of 1.2 °/min with the step width of 0.02° over 2θ ranging from 5° to 80°. Fourier-transform infrared spectroscopy (FT-IR) measurements were conducted using a Thermo Scientific Nicolet iS50 infrared spectrometer. In order to characterize the interaction between ZIF-67 and four PKU markers, the fresh sample of ZIF-67 crystals was added into the methanol solution containing phenylalanine, phenylpyruvic acid, phenylacetic acid and phenyllactic acid, respectively. The concentration of PKU markers in methanol is about 10 mM, which is employed to ensure the identifiable FT-IR signals. Then the obtained suspension was allowed for stirring at room temperature about 12 h. After centrifugation as well as removing slight PKU markers without reaction, the resulting ZIF-67/ phenylalanine, ZIF-67/phenylpyruvic acid, ZIF-67/phenylacetic acid and ZIF-67/phenyllactic acid were further used for FT-IR measurements. Fabrication of electrochemical microsensor. The microsensor was configured with threeelectrode system, i.e. working, counter and reference electrode. Both the working and counter electrodes were printed with carbon conductive ink, and the reference electrode was printed with Ag/AgCl compositions ink. All the microsenors were printed onto polyethylene terephthalate (PET) substrate. The prepared PtPd@ZIF-67 nanocomposite (5 mg) was dispersed into the mixture of ethanol (1 mL), ethylene glycol (500 L) and ethyl cellulose (3 mg) by 30 min sonication to form a homogeneous ink. After that, the obtained ink was locally deposited onto the surface of working electrode with the assistance of ink-jet printer. Electrochemical measurements. All the measurements were conducted in N2 saturated 0.1 M phosphate buffer solution (PBS, pH = 6.8) at room temperature and ambient pressure. For each detection, 8 L of test solution was dropped onto the fabricated microsensor. Electrochemical


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impedance spectroscopy (EIS) was performed in a solution containing 0.1 M KCl and 2 mM Fe(CN)63-/Fe(CN)64- and plotted in the form of complex plane diagrams (Nyquist plots) with a frequency range of 0.001 to 106 Hz. RESULTS AND DISCUSSION

Figure 1. Characterization of PtPd@ZIF-67 nanocomposite. (a) TEM image, (b) STEM image, (c) TEM image with high magnification, (d) high resolution TEM (HRTEM) image, (e) XRD patterns and (f) STEM image combined with elemental mapping results.


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Figure 1a shows the TEM image of PtPd@ZIF-67 nanocomposite, indicating the regular polyhedral shape and with side length of ~ 500 nm of ZIF-67 crystals. The STEM image in Figure 1b reveals that the synthesized ZIF-67 crystals are cubic structure. The encapsulation of PtPd NPs into ZIF-67 crystals has been demonstrated by TEM image with high magnification and HRTEM analysis shown in Figure 1c and d. It can be seen that all of the NPs are encapsulated into ZIF-67 crystals, and few of NPs are adsorbed on the outer surface of the resulting crystals. The morphology characterization of NPs is shown in Figure S1a and 1b, indicating the high-quality NPs is uniform and with size distribution of about 4-6 nm. XRD analysis is used to identify the crystal structure of synthesized sample, and the results are shown in Figure 1e. The XRD peaks generated by ZIF-67 crystals (from 5° to 38°) and the deposited PtPd NPs (broad peaks from 38° to 80°) are all observed for PtPd@ZIF-67 nanocomposite. All the sharp and prominent peaks between 5-38° match well with that of previous reported X-ray data,29 clearly indicating that pure phase of ZIF-67 are obtained. The broad peaks in the 2θ range of 38-80° exhibit higher angle shift compared with Pt standard XRD pattern (JCPDS no. 040802), indicating the decreased d-spacings and contraction of the lattice constant. The XRD peaks shift to a larger angle is due to the incorporation of smaller Pd atoms into the Pt fcc lattice, accounting for the alloy formation between Pt and Pd. Elemental analysis is further performed, and used for demonstrating the composition and distribution of PtPd NPs in ZIF-67 crystals. STEM-EDS line scan of PtPd (Figure S1c) is recorded from two NPs, the result clearly displays that both Pt and Pd L lines have the maximum concentration at the center of the particle. The similar line profiles obtained from Pt and Pd atoms verify the alloy phase of NPs, and the relatively weak signal of Pd indicates its low content in PtPd alloy NPs. The inductively coupled plasma-atomic emission spectroscopy (ICP-AES) result shows that the atomic ratio of Pt: Pd in alloy NPs is about 67: 33,


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which is consistent with the EDS line scan. A STEM image combined with selected-area element analysis mapping (Figure 1f) of Co (belonging to ZIF-67 crystals), Pt and Pd reveal that both Pt (green) and Pd (red) atoms are distributed through the whole area, revealing a homogeneous distribution of Pt and Pd in ZIF-67 crystals. All of the results verify that the resultant PtPd alloy NPs are successfully encapsulated into ZIF-67 forming the nanocomposite structure.

Figure 2. Other PtPd@ZIF-67 nanocomposites with different modified PVP amount. In our previous researches, we have found that PVP adsorbed on metal NPs surface in the reaction solution not only stabilizes the NPs, but also provides an adhesive effect for bonding metal NPs with the other materials.30,31 In the pyrrolidone ring of PVP, there are plenty of N and O atoms with lone pair electrons, which have the capability to react with metal ions through coordination interaction. We therefore speculate that there are also coordination interaction between pyrrolidone rings and Co2+ during the heterogeneous nucleation and growth process of ZIF-67. In the synthesis system, the PVP amount has an important influence for the morphology of obtained nanocomposites. With three more times washing the NPs, there is less residual PVP on the NPs surface, resulting in particle aggregation inside of ZIF-67 crystals (Figure 2a). In


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addition, a lot of nanoparticle clusters are formed on the outer surface of ZIF-67 when excess free PVP (50 mM) is introduced in the reaction solution (Figure 2b). By changing the NPs and controlling PVP amount, other PtIr@ZIF-67 nanocomposite has also been prepared (data are not shown here). It demonstrates that the developed encapsulation procedure could be extended to the synthesis of other nanocomposite analogues. After the synthesis of composite material, the sample washing and electrochemical pretreatment procedures have been carried out to avoid the side effect of PVP on the following electrochemical detection.

Figure 3. (a-b) The configuration of fabricated microsensor (WE = working electrode, CE=counter electrode, RE=reference electrode), (c) Nyquist plots of blank GCE, blank microsensor, ZIF-67 modified microsensor and PtPd@ZIF-67 modified microsensor (the equivalent circuit used to fit the Nyquist plots is shown in inset, RΩ and Rct represent electrolyte resistance and charge transfer resistance, C is the double layer capacitance, Ws corresponds to Warburg impedance), (d) metabolic pathways of phenylalanine in PKU patients.


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Next, the synthesized PtPd@ZIF-67 nanocomposite is prepared into printable ink and locally deposited onto the working electrode of microsensor. The configuration of fabricated microsensor is shown in Figure 3a and 3b. The size of each microsensor is about 8 mm  38 mm, the diameter of working electrode is 1.2 mm, and the distance between working electrode and the other two electrodes is 0.6 mm, respectively. This biosensor could accommodate 8 L of test sample. Generally, EIS could be used to estimate the interfacial properties of electrode.32 The depressed semicircle at high frequency is related to electron transfer resistance (Rct) at the electrode surface. From the Nyquist plots in Figure 3c, a visible depressed semicircle is observed for blank glassy carbon electrode (GCE), while the ESI of blank microsensor displayed a smaller semicircle, indicating fast electron transfer capability. After ZIF-67 crystals deposition, Rct is decreased from 59.37 to 38.32 Ω. The Rct of microsensor modified with PtPd@ZIF-67 nanocomposite is further decreased to 26.43 Ω, demonstrating a great possibility for electrochemical applications. Herein, the PtPd@ZIF-67 modified microsensor is employed for measurements of PKU markers. According to pathogenesis, the deficiency of PAH is observed in PKU patients, then the conversion of phenylalanine to tyrosine is blocked and another metabolic pathway is incurred.33 This causes the elevated levels of phenylalanine in blood and phenylpyruvic acid, phenylacetic acid and phenyllactic acid in urine. The metabolic pathways of phenylalanine are shown in Figure 3d. Accordingly, phenylalanine, phenylpyruvic acid, phenylacetic acid and phenyllactic acid are often used as markers for the diagnosis of PKU.


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Figure 4. FT-IR spectra demonstrating the interaction between ZIF-67 and four different PKU markers. (a) phenylpyruvic acid, (b) phenylacetic acid, (c) phenyllactic acid and (d) phenylalanine. Since ZIF crystals are consisted of tetrahedral metal centers connected by bent imidazolatederived organic ligands, one of the characteristics is to selectively react with certain molecules by the easy formation of chemical bonds through their imidazolate linkers. Then, FT-IR measurements are performed to demonstrate the interaction between ZIF-67 crystals and different PKU markers, and the results are shown in Figure 4. In the spectrum of pure ZIF-67, the bands in the spectral region of 600-1340 cm-1 and 1340-1500 cm-1 are associated with the plane bending and stretching of entire imidazole ring, respectively.34 The C-N stretching


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vibration in imidazole ring is appeared at 1578 cm−1, and peaks for the aromatic and aliphatic CH stretches were expected at 2927 cm−1 and 3136 cm−1.35 All of the FT-IR bands in the spectrum of ZIF-67 are in accordance with results reported in previous literatures.36,37

Scheme 1. Two possible interactions between the ligand (2-methylimidazole) of ZIF-67 crystals and carboxyl group in PKU markers. After introduction of phenylpyruvic acid to ZIF crystals, a strong band and two weak bands are emerged in the spectrum of ZIF-67/phenylpyruvic acid sample (shown in Figure 4a). In the chemical structure of phenylpyruvic acid, there is only carboxyl group having the possibility to react with imidazole framework of ZIF-67 crystals (Scheme 1). The possible interactions between ZIF-67 and phenylpyruvic acid are formation of N-HO hydrogen bond or acylation of nitrogen atom in imidazole ring, respectively.38,39 The detailed reaction is shown in Scheme 1. According to the spectrum of ZIF-67/phenylpyruvic acid, the strong bands at 1663m-1 is related to the stretching vibration of CONH or C=O, while the two weak bands in 3150-3350 cm-1 rang


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are generated by CONH.40 In addition, the broad hydroxyl stretching vibrations have not been observed in the spectrum of ZIF-67/phenylpyruvic acid. All of these results could deduce that there is an acylation reaction in imidazole ring of ZIF-67 in which nitrogen atom is acylated by carboxyl in phenylpyruvic acid. The interaction of ZIF-67 with other three PKU markers is also measured with FT-IR spectrometer, respectively. From Figure 4b, a similar strong peak at about 1689 cm-1 is arisen for ZIF-67/phenylacetic acid, and there is also no broad hydroxyl stretching vibrations shown up. The results obtained for phenylacetic acid verify the proposed acylation reaction between ZIF-67 and carboxyl group. We also noted that there is carboxyl group in other two PKU markers; however, nothing happens for phenyllactic acid and phenylalanine interacting with ZIF-67 crystals. From Figure 4c and 4d, the FT-IR spectra of ZIF-67/phenyllactic acid and ZIF67/phenylalanine display the almost identical result as for pure ZIF-67 crystals, respectively. We carefully checked the chemical structures of these four PKU markers shown in Figure 5, the same carboxyl group and characteristic property of phenyl group are coexisted in phenylpyruvic acid, phenylacetic acid, phenyllactic acid and phenylalanine. The disparity of these four PKU markers is the different functional groups attached to first (alpha-) carbon atom of carboxyl group. For the acylation reaction between 2-methylimidazole linker and carboxyl group, nitrogen atom with lone-pair electrons is subjected to the electrophilic attack from carbon atom with partial positive charge (δ+) in the carbonyl group, and forming an active complex as transition state.41 Subsequently, the carboxamide (N-C=O) compound is produced through the removal of water (H2O). In the reaction, the chemical structure of acylating agents affects greatly on the activity to electrophilic attack and further result in different reaction efficiency.42 In particular, the attached functional group on alpha- carbon atom of carbonyl group is closely


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related to the capability for electrophilic attack.43 In the structure of phenylpyruvic acid, an electron withdrawing group of carbonyl group is located on the alpha-site of carboxyl group, while the electron donating group is appeared on the same position for phenyllactic acid and phenylalanine. In addition, there is no functional group attached to the alpha- carbon atom of carboxyl group in the structure of phenylacetic acid. The electron withdrawing group would active carboxyl group and further facilitates the reaction, while the electron donating group actually do the reverse. Accordingly, the disparity on the structure of PKU markers endows them different activity interacted with ZIF-67.

Figure 5. Responses of carbon material, ZIF-67 crystals, and PtPd@ZIF-67 modified microsensors towards PKU markers as well as some common amino acids. We next evaluate our proposed acylation reaction induced sensing of phenylpyruvic acid and phenylacetic acid over PtPd@ZIF-67 modified microsensor. Firstly, four PKU markers as well as some common amino acids with same concentration of 200 nM are measured, the


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performance of pure ZIF-67 crystals and carbon material modified microsensors are also included. From Figure 5, the visible responses towards phenylpyruvic acid and phenylacetic acid are observed. Despite with similar chemical structure, not apparent response signals are produced by other measured species. In addition, PtPd@ZIF-67 nanocomposite presents clear superiority over pure ZIF-67 crystals and carbon material. For pure ZIF-67 crystals, the similar but lower response signals are obtained for all measured substances. And carbon material almost has no response towards PKU markers and amino acids. These results make clear that ZIF-67 crystals has the capability of capture PKU markers through acylation reaction, and the encapsulated PtPd alloy nanocrystals further enhance electron conductivity of the microelectrode surface. Moreover, the chemical structural characteristics of phenylpyruvic acid and phenylacetic acid give them the particular activity over the other two PKU markers to interact with ZIF-67 crystals. The sensing performances of PtPd@ZIF-67 modified microsensor towards phenylpyruvic acid and phenylacetic acid are then evaluated in terms of sensitivity, detection limit and selectivity. The optimum test conditions are at the temperature of 25 °C and pH value of 6.8, which is based on the results shown in Figure S2. The CV responses of PtPd@ZIF-67 modified microsensor towards the two markers are shown in Figure S3, and the measurements over GCE, blank microsensor, pure ZIF-67 modified microsensor are also included for comparison. The blank GCE has slight responses to phenylpyruvic acid and phenylacetic acid only if the voltage is higher than 0.4 V (Figure S3a), while the onset potential over blank microsensor is lowered to about 0.2 V (Figure S3b). After modification with ZIF-67 crystals, the onset potential is further reduced to about 0.1 V, and much higher response signal is produced (Figure S3c), which could be ascribed to the selective capture capability of ZIF-67 material. Then again, the encapsulated PtPd NPs provide fast electron transfer ability and high chemical activity, which work


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synergistically with ZIF-67 crystals so that the composite material can bring superiority than either one of them. From Figure S3d, the response signals generated from PtPd@ZIF-67 are much higher than that of pure ZIF-67. In addition, the onset potential of PtPd@ZIF-67 modified microsensor to the two markers is very low. The clear response signals could be observed even at the potential of 0 V. In order to acquire the detectable signal and avoid the interference from other electrochemical active species, the applied potential for the following amperometric measurements is chose as 0.1 V. According to literatures, the urinary content of phenylpyruvic acid in healthy newborns is about several M, and phenylacetic acid usually has a higher level than that of phenylpyruvic acid.44,45 The abnormal accumulation of these markers in PKU patients is usually about ten times or even much higher than normal levels.9,45 Thus, the measurements of phenylpyruvic acid and phenylacetic acid over physiological range have been conducted. From Figure 6a and 6b, the amperometric detection of phenylpyruvic acid displays the linear signal increase with gradually elevating the concentration. The observed stepwise current increase indicates PtPd@ZIF-67 modified microsensor has the ability to sensing phenylpyruvic acid from nM to μM. The different linear relationships in high concentration and low concentration may be attributed to variations in molecular diffusion rates of different concentration ranges. The detection limit measurement (Figure 6c) shows that the developed PtPd@ZIF-67 modified microsensor could detect phenylpyruvic acid even the concentration as low as 0.1 nM. Figure 6d demonstrates that PtPd@ZIF-67 modified microsensor also could be used for detection of phenylacetic acid with good linear relationship (R2=0.9823) from 5 to 500 M. The detection limit of developed microsensor for phenylacetic acid is estimated as 20 nM (Figure 6e). In addition, several electrochemical active species contained in body fluid have been included for selectivity


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evaluation. As shown in Figure 6f, there is no obvious current responses with the addition of these physiologically compounds or ions, and the responses to 5 M of phenylacetic acid and phenylpyruvic acid are well recurred in the relatively complex environment. Accordingly, we emphasize that such a highly selective response to PKU markers is obtained over the explored PtPd@ZIF-67 modified microsensor, without using of selective membranes or bio-recognizers.

Figure 6. Electrochemical sensing performance of PtPd@ZIF-67 modified microsensor towards phenylpyruvic acid and phenylacetic acid. (a) Current responses for successive addition of phenylpyruvic acid from 1 to 200 M, (b) responses to phenylpyruvic acid in the concentration range of 20 nM to 1 M, (c) detection limit evaluation for phenylpyruvic acid, (d) responses to phenylacetic acid in the concentration range of 5 to 500 M, (e) detection limit measurement for phenylacetic acid, (f) selectivity evaluation to PKU markers and some potential interferences (urea, uric acid (UA), glucose, creatinine (CRE), NaNO3 and KCl).


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Figure 7. The repeatability and stability (inset) evaluation of PtPd@ZIF-67 modified microsensor. The concentration of phenylacetic acid and phenylpyruvic acid for repeatability measurements is 5 μM. The repeatability and stability evaluation of PtPd@ZIF-67 modified microsensor have also been performed, and the results are shown in Figure 7. The developed microsensor displays the good repeatability with a standard deviation of 2.86 for phenylacetic acid detection and a standard deviation of 5.74 for phenylpyruvic acid measurement, respectively. And the stability of the microsensor is satisfied, the current of microsensor measured in PBS keeps about 81% of initial value after ten days of testing. The comparison of sensing performances in terms of detection strategy, linear range and limit of detection (LOD) with that of other sensing materials reported in recent publications is shown in Table S1. From the comparison, our developed PtPd@ZIF-67 modified microsensor shows the features of easy to use, simple and no need to care about the activity of bio-recognizers.13,46,47 In addition, low LOD and wide linear response range make the developed microsensor with superiority than that of other methods.48,49


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For the diagnosis of PKU in patients, all the markers are usually co-existent in body fluids. It is hard to distinguish phenylpyruvic acid and phenylacetic acid if only from the produced current signal. According to previous reports, the cooperative abnormal accumulation of phenylpyruvic acid and other markers could be taken as the initial diagnosis proof of PKU.45,46 Therefore, we could measure the total expression of these markers as early warning or evaluation of therapeutic effect. It also could build a quantification model for the analysis of these markers. The metabolism in PKU patients indicates that phenylacetic acid is converted from phenylpyruvic acid, and the cut-off value of phenylacetic acid is almost twice as much as that of phenylpyruvic acid.44 Taking into account the roles played by them in metabolic pathway as well as the relationship between their levels in body fluids, and then the quantitative analysis of their mixture is possible.

CONCLUSIONS In summary, a PtPd@ZIF-67 modified microsensor was developed and used for the measurement of PKU markers. Taking the advantages of ZIF-67 crystals, the chemoselectivity towards PKU markers was achieved, and the interaction between ZIF-67 and PKU markers has been demonstrated by FT-IR spectra. An acylation reaction between imidazole linker of ZIF-67 and carboxyl in PKU markers is responsible for the sensing mechanism. After encapsulation of PtPd alloy NPs into ZIF-67 crystals, the electron transfer capability and corresponding electrochemical performance were further elevated. In addition, the developed PtPd@ZIF-67 modified microsensor also could distinguish PKU markers with other amino acids analogues and some physiologically compounds or ions, indicating a good anti-interference ability. These results demonstrated that PtPd@ZIF-67 modified microsensor is promising for screening of PKU.


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Corresponding Author * E-mail: [email protected]; Tel: +86-21-69982225. Author Contributions ⊥X.X.

and D.J. contributed equally to this work.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This research is supported by National Key R&D Program of China (2016YFA0200800), National Natural Science Foundation of China (61604163, 61527818), NSF of Shanghai (17ZR1410000), Key Research Program of Frontier Sciences of Chinese Academy of Sciences (QYZDJ-SSW-JSC001). We also thank Dr. Pengfei Hu for the characterization of synthesized materials. REFERENCES 1.

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Detection of Phenylketonuria Markers Using a ZIF-67 Encapsulated

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