Microneedle pH Sensor: Direct, Label-free, Real Time Detection of

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Microneedle pH Sensor: Direct, Label-free, Real Time Detection of Cerebrospinal Fluid and Bladder pH Ganesh Kumar Mani, Kousei Miyakoda, Asuka Saito, Yutaka Yasoda, Kagemasa Kajiwara, Minoru Kimura, and Kazuyoshi Tsuchiya ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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ACS Applied Materials & Interfaces

Microneedle pH Sensor: Direct, Label-free, Real Time Detection of Cerebrospinal Fluid and Bladder pH Ganesh Kumar Mani1,*, Kousei Miyakoda2, Asuka Saito2, Yutaka Yasoda2, Kagemasa Kajiwara3, Minoru Kimura3 and Kazuyoshi Tsuchiya1,4 1

Micro / Nano Technology Center, Tokai University, 4-1-1 Kitakaname, Hiratsuka,

Kanagawa, 259-1292, Japan. 2

Graduate School of Science and Technology, Tokai University, 4-1-1 Kitakaname,

Hiratsuka, Kanagawa, 259-1292, Japan. 3

Department of Molecular Life Science, Tokai University, 143 Shimokasuya, Isehara-

shi, Kanagawa 259-1193, Japan. 4

Department of Precision Engineering, Tokai University, 4-1-1 Kitakaname, Hiratsuka,

Kanagawa, 259-1292, Japan.

---------------------------------------------------------------------------------------------------------*Corresponding Author Dr. Ganesh Kumar Mani Ph.D. Micro / Nano Technology Center Tokai University Shonan Campus, Building No: 12 (First Floor) 4-1-1 Kitakaname, Hiratsuka, Kanagawa, 259-1292, Japan. Tel: +81 463 58 1211 Ex: 4791 Fax: +81 0463 50 2480 [email protected]; [email protected] (Ganesh Kumar Mani) [email protected] (Kazuyoshi Tsuchiya) 1 ACS Paragon Plus Environment


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Abstract Acid–base homeostasis (body pH) inside the body is precisely controlled by kidney, lungs and buffer systems, even a minor pH change could severely affect many organs. Blood and urine pH tests are common in day-to-day clinical trials without much effort for diagnosis. Always there is great demand for in vivo testing to understand more about body metabolism to provide effective diagnosis and therapy. In this communication, we report the simple fabrication of microneedle based direct, label free and real time pH sensor. The reference and working electrodes were Ag/AgCl thick film and ZnO thin film on tungsten (W) microneedle respectively. Morphological and structural characteristics of microneedles were carefully investigated through various analytical methods. The developed sensor exhibited the Nernstian response of -46 mV/pH. Different conditions were used to test the sensor to confirm their accuracy and stability such as various buffer solutions with respect to time and compared the reading with commercial pH electrodes. Besides that, fabricated microneedle sensor ability is proven by in vivo testing in mice cerebrospinal fluid (CSF) and bladder. The pH sensor reported here is totally reversible and results were reproducible after several routine testing.

Keywords: pH; Microneedle; ZnO; Sensor; Cerebrospinal fluid; Mice; in vivo.

2 ACS Paragon Plus Environment

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Introduction Accurate quantification of pH is of great importance in many biological processes and disease diagnosis. Blood plasma, intestinal fluid and CSF are most sensitive to acid-base balance. Changes in simple ventilatory rate affect blood arterial, gastrointestinal tract and cerebrospinal fluid pH within seconds.1–3 Even neuronal activity accompanied by considerable changes in pH in brain.4,5 Mechanism underlying this pH change are not completely understood till date. In addition to the understanding of acid–base homeostasis in our body, analysis of real time CSF pH also offers key insight into the status of the central nervous system (CNS) such as Alzheimer’s, meningitis, multiple sclerosis, and CNS tumors. In such cases, real time pH monitoring would help to avoid acidosis or alkalosis and gain more insight to therapeutic strategies with high spatial resolution, accurate and precision. Typically, pH monitoring is made through the use of pH test strips, metal/glass electrodes, imaging and spectroscopic techniques. But, for decades the search of reliable in-vivo pH monitoring has never stopped. Cost effectiveness, ease of fabrication, reliability, biocompatibility and miniaturization are the critical parameters of in-vivo testing of pH. A large number of protocols have been demonstrated to analyze in-vivo pH monitoring such as luminescence imaging6, fiber optic probe7, near-infrared reflectance spectroscopy 8, absorption / fluorescence spectroscopy 9, magnetic resonance spectroscopy

10

, etc.

Among all, high sensitivity fluorescence spectroscopy is widely used as it is harmful to the cells. However, many factors including optical path length, changes in temperature, excitation intensities will affect the fluorescence based sensors ability. Moreover, the drawbacks of the fluorescence dyes in in-vivo imaging may led to certain inevitable side effects.11



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Hence, to overcome to these limitations, recently microneedle type sensors gains more attention in contrast to conventional planar type solid state electrodes due to its ability to penetrate the samples painlessly due to fewer interaction with nerves than the conventional hypodermic needles. As perforation takes only few seconds with microneedles, measurement and analysis could be quite simple. Besides that, the small dimensions of the sensor could be used for more precise pH measurements in controlled environments such as brain fluid, intestine and bladder without taking out the samples externally.12 In recent days microneedle type sensors fabricated in number of ways like laser micromachining, selective electrodeposition13, reactive ion / wet / dry etching, laser ablation, laser cutting, etc.14 So far most of the microneedles based on various nanostructures have been developed for drug delivery and glucose sensors. Rather very limited attention was given to the application of pH sensors. However, recent works shows that the microneedles based electrodes can be used for pH detection too. Choi et. al developed needle type pH sensor by selective electrodeposition and used iridium oxide as the sensing element13. Also boron doped diamond microelectrode developed by Fierro et. al could be used to identify stomach disorders.

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These fabrication methods

exhibit number of inherent disadvantages such as poor reproducibility, difficulty in making contacts, fragility, etc. To overcome the limitations associated with the available technologies, here we report the microneedle based direct, label free real time pH monitoring of the body fluids. We reported the first demonstration of the ZnO and Ag/AgCl based microneedle electrode for real time pH monitoring and successfully tested in mice CSF and bladder. Numerous studies of various metal oxides as pH sensitive materials like WO316,17, MnO18, RuO219, Ir2O320, ZnO21–24 have been reported in last few decades.


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Well known amphoteric property, high crystallinity, hardness, biocompatibility, chemical stability, large exciton band energy, polar and non-polar property makes ZnO is one of the best candidates for pH sensing element

25–33

. Although there are previous

examples on ZnO (nanowalls, nanowires, nanorods) as pH sensor22,21,34 are successful in sensing various pH ranges. So far, many research groups have been focused only on improving the sensing performances. To date, various types of pH sensors fabricated based on ZnO like resistive35,36, potentiometric37,38, extended gate field effect transistor39 and surface acoustic wave40 based devices. However, combination of highly pH sensitive material with microneedles shows potential in exploring a new kind of sensors which can be highly supportive in biomedical field. In particular, W widely adapted in making surgical needle/knives owing to its high Young’s modulus. Morphological and structural characteristics of microneedles were investigated through X-Ray Diffraction (XRD), Scanning Electron Microscope (SEM), X-Ray Photoelectron Spectroscope (XPS) and High Resolution Transmission Electron Microscope (HRTEM). The overall merits of the developed microneedle based pH sensors may have broad applications in Bio-MEMS technologies.

Methods Materials 300 µm diameter W microneedles with the purity of 99.95% were directly purchased from Nilaco Corporation, Japan. ZnO target (99.95% purilty, 60 mm in diameter) were obtained from Furuuchi Chemical Corporation, Japan for deposition of ZnO thin films on microneedle. Ag/AgCl ink was obtained from ALS Co., Ltd, Japan. The portable pH meter was calibrated with standard buffered solutions (pH 4.06 & pH



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6.86) obtained from HORIBA Advanced Techno Co., Ltd., Japan to validate the readings from the developed microneedle pH sensor. Ultrapure water from Milli-Q System (Millipore, USA) was used throughout the experiment. All products were analytical grade and were used without any further purification.

Sensing Electrode Preparation W microneedle substrate (30 mm length; 300 µm diameter) were initially cleaned with ethanol and deionized water to remove the surface contaminants. Then, 200 nm ZnO thin films were deposited on W microneedle by RF magnetron sputtering. Prior to deposition to get uniform ZnO deposition on W microneedle, W microneedle was fixed in a motor, which will rotate 25 rpm perpendicular to the target source. ZnO was deposited around 5 mm in length of the microneedles and rest places were masked with Kapton tape. The deposition conditions such as sputtering power, Ar flow rate, working pressure and sputtering time were fixed as 100 W, 50 sccm, 1 Pa and 30 min respectively. For reference electrode, W microneedle was dipped about 5 mm in Ag/AgCl ink and dried overnight in ambient atmosphere. Finally copper wire was welded with W microneedles for pH sensing measurements. The complete fabrication and testing scheme is shown in Figure 1.

Characterizations & In vivo pH Sensing Studies The structural studies were characterized using X-Ray Diffractometer (XRD) (LabX XRD 6100, Shimadzu, Japan) and X-Ray Photoelectron Spectroscope (XPS) (PHI Quantum 2000, Physical Electronics, Inc., USA). Morphological studies were analyzed using Scanning Electron Microscope (FE-SEM) (JEOL, JCM 6000, Japan).


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Field Emission High Resolution Transmission Electron Microscope (FE-HRTEM) images were obtained from Hitachi HF 2200 TU, Japan at an accelerating voltage of 200kV.

Animal Experiments A preliminary pH testing was done with standard buffer solutions. pH sensing performances were recorded by 2325 Bipotentiostat from ALS Co., Ltd., Japan. In-vivo testing was performed in brain and bladder of the healthy mice in order to check the capability of the developed microneedle pH sensor in real time. Female C57BL/6J mice supplied from CLEA Japan (10 weeks old) were used for experiments. These mice were maintained in a cage (200 mm × 300 mm) under the specific pathogen free conditions with a 12 hour light-dark cycle before experiments. Food pellets and water were freely available before anesthesia. Following induction of anesthesia with 5% isoflurane in air, the mice were placed on a heating blanket to keep their body temperature and maintained with 2~2.5% isoflurane in air. Surgeries were performed in neck and abdomen of the mice with stereo microscope under white light illumination. The measurements were performed on 5 control mice in order to evaluate the stability of the microneedle. For cerebrospinal fluid testing, an incision around 2 cm was made on top of the head.

After separating the skin, muscle and veins around occipital region,

exhibited dura master between occipital bone and first cervical vertebrae was cut carefully, and the microneedle was gently inserted about 2 mm in to the brain to measure the real time pH values. Continuous measurements were made throughout the process of by microneedle penetration. After sensing the pH of cerebrospinal fluid, the urinary bladder was exposed and the pH-sensing microneedle was inserted. After each



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pH testing, the microneedle was rinsed well in doubly deionized water. Further, 33 gauge needle attached with polyurethane tube was inserted into brain and collected CSF externally under gentle aspiration to validate the pH values with commercial pH meter. Excess care was taken to avoid blood contamination. Samples in the syringe quickly transferred to commercial pH meter for validation. The same process was followed to investigate the bladder pH. This animal study was conducted in accordance with guidelines for the care and use of animals for scientific purposes at Tokai University and approved by the Institutional Review Board of Tokai University School of Medicine (Permit Number: 161075).

Results and Discussion XRD patterns of bare W microneedle, ZnO on W microneedle and Ag/AgCl ink on W microneedle was shown in Fig. 2 (a). W microneedle showed peak at 2θ = 40, 58, 73° due to reflection from the (110), (200), (211) crystal planes and no other impurity peak was detected. The W microneedle exhibited (110) preferential orientation and it corresponds to body - centered cubic crystal structure41 and peaks were matched and indexed according to the standard X-ray powder diffraction pattern no: 04-080642. XRD pattern of ZnO thin film coated over the W microneedle showed peak at 2θ = 34° which corresponds to (002) plane according to JCPDS card no: 36-1451. Only (002) crystal plane was observed corresponds to ZnO and remaining peaks was corresponds to the W microneedle (underlying layer). The (002) plane indicates that the formation of highly textured c-axis oriented ZnO film ascribed to hexagonal wurtzite structure and broadening of peak indicating smaller crystalline size. XRD patterns of Ag/AgCl ink coated on W microneedle showed the cubic phase formation of AgCl crystals and peaks


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at 2θ = 27, 32, 46, 55, 57, 67°, were ascribed to planes (111), (200), (220), (311), (222) respectively (JCPDS No. 31-1238). The metallic Ag peaks are also detected at 2θ = 38, 44, 64 and 77° were ascribed to (111), (200), (220) and (311) reflection planes (JCPDS No. 65-2871)43. In the case of Ag/Agcl ink no peak related to W microneedle (underlying substrate) were detected it is attributed that the higher thickness of Ag/AgCl. Fig. 2 (b-d) shows the higher resolution XPS Survey scan of Zn 2p and O1s core-level region. Peaks in the survey scan are assigned to Zn, O and no peak of other element were noticed which indicates the pure phase formation of ZnO thin film. From, Zn 2p spectra we observed two distinguish peaks located at 1019.76 and 1042.30 eV corresponds to Zn 2p 1/2 and Zn 2p 3/2 respectively. The peak at Zn 2p spectra is attributed to the spin-orbit splitting and binding energy difference between Zn 2p 1/2 and Zn 2p 3/2 is 23 eV, which is in good agreement with energy splitting of ZnO.44 O1s core-level signals was deconvoluted and fitted with Gaussian function. It resolved in to two peaks with binding energies of 528.68 and 530.24 eV respectively. The peak at 528.64 eV may be attributed to O2- ions adsorbed on the surface of the film from the atmosphere. Peak at 530.24 eV may be related to the lattice O2- ions in the Zn-O bonding of the hexagonal wurtzite structure.45,46 Fig. 3 (a & b) shows the HRTEM and selected area electron diffraction pattern (SAED) of the ZnO. HRTEM analysis confirms the crystalline nature and spherical shape of the ZnO. The observed size distribution of ZnO found to be in the range of 20 – 30 nm. Fig. 3 (b) shows the interfacial fringes pattern of ZnO, the distance between two adjacent lattice planes

are 0.26, 0.28, 0.52, 0.19 nm

corresponds to (002), (100), (001), (102) wurtzite crystal plane respectively. The corresponding SAED pattern of is shown in the inset of Fig. 3 (b) exhibits number of



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Debye rings each ring is indexed to (002), (100), (101), (102), (110), (103) and (200) crystallographic planes, which attributed to polycrystalline nature which is in consistent with the XRD results [1]. SEM micrograph of Ag/AgCl and ZnO on W microneedle is shown in Fig. 4. The diameter after deposition of Ag/AgCl (Fig. 4 (a & b) and ZnO (Fig. 4 (c & d)) on W microneedle was found to be 305 and 302 µm respectively. The higher magnification SEM image reveals that the monotonous distribution of ZnO and Ag/AgCl on the surface of microneedle, no aggregations and visible defects were noticed. Microneedle coated on Ag/AgCl exhibits smooth, spherical like morphology and porous in nature, whereas, ZnO coated microneedle exhibit smooth surface with more compact in nature and no distinguish grains and grain boundaries were observed. To validate the pH sensing ability of the fabricated ZnO-Ag/AgCl microneedle, we have carried out sensing performances at room temperature by potentiometric method using standard pH buffer solutions as well as in vivo testing in mice. For ease and handling, copper wire was bonded between W microneedle and potentiostat. As shown in the schematic, copper connection area or bare W was not immersed in the solution to observe, the current changes only from the ZnO-Ag/AgCl electrodes. The electrodes were thoroughly cleaned with deionized water before each pH measurements. The pH test was repeated with fresh pH buffer solutions at least 5 times and sensitivity was calculated. Fig. 5 (a) shows time dependent open circuit potential of the electrodes in various buffer solutions from 1.68 to 9.18. The open circuit potential was measured immediately after immersing the microneedles into the buffer solutions. The magnitude of the open circuit potential can be easily related with pH values. The pH microneedle sensor produced in this work exhibited the mean sensitivity of -46.35 mV/pH (Fig. 5


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(b)). pH sensitivity for commercial Ag/AgCl electrode with ZnO microneedle was measured for comparison. The sensitivity was found to be -46.35 mV/pH and -43.42 mV/pH for Ag/AgCl microneedle electrode and commercial Ag/AgCl electrode respectively. It clearly revealed that Ag/AgCl ink based microneedle showed slightly higher sensitivity. But the change between Ag/AgCl ink and commercial Ag/AgCl electrode was small. Also the measured potential shows small standard deviation which confirms that the experimental method used in this study is stable and reproducible. Furthermore, dynamic pH sensing (Fig. 5 (c)) was also performed by sequentially switched between various pH solutions. Initial sensor stabilization was found to be 14 s. No change in open circuit potential was not seen upon immersing the microneedles for 1 hour in buffer solutions. Noise was minimal during measurements typically in the range of ± 5 mV. Further to evaluate in vivo pH sensing performance, mice CSF and bladder pH was measured using microneedle. Photo image of the in vivo mice CSF and bladder pH experimental set-up was shown in Fig. 6 (a-d). The measurement time was limited to 60 s to minimize the pain. Mice CSF and bladder pH measurement results are shown in Fig. 7 (a & b). The stabilization time of the sensor in CSF and bladder was 18 s and 30 s respectively. And the identified values via. microneedles were pH 7.45 and pH 6.30. The test was conducted individually in 5 control mice on different days and the results are as shown in Fig. 7 (c). The average estimated values of 5 different control mice were pH 7.34 and pH 6.4 in CSF and bladder respectively. Moreover, to check the sensitivity of the developed microneedle pH sensor data was compared with commercial pH sensor results. It must be noted that, it is very difficult to find the theoretical pH data of the mice CSF and bladder due to neuronal activity changes the localized pH via. various



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mechanisms. Hence, the CSF and bladder urine were collected externally under the same circumstances and evaluated. Firstly, CSF collected in polystyrene container (about 0.3 µL) and transferred into portable pH probe within 45 s. The pH results obtained by commercial pH meter exhibited slightly higher pH values than measured by microneedles (Fig. 7 (d)). Removal of body fluids from their buffered environment obviously can effect in their pH. But near physiological values achieved in both cases. When ZnO surface contacts with the electrolyte solution, H+ bonding sites in ZnO gets hydrogenated and build change in the surface charge. This hydrogenated sites can protonate or deprotonate depends on the electrolyte solution. The electrode constructed in this work is Ag | AgCl | Cl- is a reference electrode against ZnO as working electrode. The overall potential cell model developed is (Eqn. 1), W | ZnO | Test Electrolyte Cl- | AgCl | W − − − − − − (1) According to Nernst equation

34

, the ideal pH electrode potential can be denoted as

(Eqn.2), = −

.

− − − − − −(2)

Where, E0 is the standard ZnO electrode potential,

R is the universal gas constant

(8.314 J/mol K), T is the absolute temperature (298 K) and F is the Faraday constant (96487.3415 C mol-1). At room temperature Nernst equation give a slope value of 59.1 mV/pH. The probable acid and base reactions (Eqn. 3 & 4) on ZnO surface is given below 47, For acid, + 2 + → () − − − − − −(3) For base, + 2 + 3 → ( ) − − − − − −(4) For more adsorption / desorption process the unsaturated dangling bonds at the surface usually compensated by reactive molecules such as H2O.48 According to the above 12 ACS Paragon Plus Environment

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mentioned results, pH measurements were done using various buffer solutions as well as in vivo biological mediums with high accuracy and high degree of linearity. In buffer solution, microneedles exhibited high stability once immersed in solution. At the same time, in CSF and bladder it may take longer time to get stability than stable buffer solution due to interaction with many biological molecules. Also, it is known fact from Wang et. al research that CSF replenishes rapidly at ~ 3.3 x 10-4 ml/min in small mammalian species like mice, although measuring pH using the available total CSF around ~36 µL is perhaps most difficult task

49–51

. Even extracellular pH changes was

measured in rat hippocampal slices by pH sensitive dyes previously by phenol red and found that even a small change in neuronal activity also accompanied by considerable change in pH 4. A table of current and past in-vitro & in-vivo pH sensing studies presented in Table 1. Long time exposure at higher pH levels cause damage to the microneedle pH sensor. We attempted to to measure higher pH. But the observed values is unstable due to the deposited layer is thin and can be easily broken when exposed to long time. Also for the normal function of our body pH range is 6.9 to 7.4. So we believe that the developed sensor can measure the pH range from 2 to 9 is good enough to perform clinical studies. Furthermore this kind of electrode can work well in various environments due to amphoteric properties of ZnO. The experimental set-up can easily adapted in clinical laboratories by simply placing it on the desired area of interest. Sensitivity of our sensor remains constant during 3 months of repeated usage. Further work is needed to refine our methods in various animals and human CSF. The success of such work is would lead to the application of this technology to human clinical trials.



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Conclusions In this study, we demonstrated the pH sensing measurements utilizing W based microneedles. In-vivo mice CSF and urine was investigated in this study to prove the capability of the developed microneedle sensor. Microneedles exhibited the sensitivity around -46.35 mV/pH. Even though the developed sensor exhibited lower than ideal Nernstian response, this is the promising step towards the development of pH sensor which can be used either during surgery or after for diagnosis as well as better understanding of the metabolic processes. The microelectrode technology reported in this present work could lead to a new generation of diagnostic tools for detection of various biological analytes simultaneously in the near future.

In future, various

customized microneedles will be developed for various analytes. As a future goal towards creating microneedle pH sensor with more improved properties, puncturing pressure, force needed to pierce tissues or cells, friction co-efficient between needle and cells will be evaluated theoretically as well as experimentally.


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Acknowledgements Authors wish to express their sincere thanks to the Micro/Nano Technology Center (MNTC), Tokai University (Shonan Campus), Japan for their infrastructural and financial support. Authors express their sincere thanks to Tokai Imaging Center for Advanced Research (TICAR), Tokai University (Shonan campus), Japan for additional characterization techniques. We would also like to show our gratitude Ms. Iwao, Department of Laboratory Animal Science, Support Center for Medical Research and Education, Tokai University for assisting animal experiments.



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Acupuncture

Needle

and

Its

Neurotransmitters. Sci. Rep. 2015, 5 (January), 11627 (1-9).


Application

in

Detecting

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1 Schematic of complete fabrication and testing of microneedle pH sensor.



Ag AgCl ZnO W

Zn 2p1/2 Zn 2s

O Auger

100000

0

Zn Auger

Zn 3d Zn 3p Zn 3s

200

(211)

(110)

(200)

200000

Zn Auger

Zn Auger

300000

Zn Auger

400000

O 1s

(311) (211)

(200)

(110)

Intensity (a.u.)

400

(b)

600000 500000

(220)

(311)

(200) (220)

600 (002)

Intensity (a.u.)

800

(200)

(111)

1000

Ag/AgCl ZnO W

(222)

(111)

(a)

Zn 2p3/2

1200

0 20

25

30

35

40

45

50

55

60

65

70

75

0

80

200

400

2θ (deg.)

1000

1200

1400

(d)

O 1s

22000 20000

Intensity (a.u.)

60000 1019.75 eV

Zn 2p1/2

50000

800

24000

(c)

Zn 2p3/2

70000

600

Binding Energy (eV)

80000

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 31

40000 30000

1042.80 eV

528.56 eV

18000 16000 14000 530.24 eV

12000

20000

10000

10000 0 1010

8000 1015

1020

1025

1030

1035

1040

1045

1050

526

528

530

532

534

536

Binding Energy (eV)

Binding Energy (eV)

Figure 2 a) XRD pattern of W, ZnO & Ag/AgCl. XPS spectra of ZnO thin film b) survey spectra, c) Zn 2p and d) O 1 s spectra.


538

540

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3 a) Low magnification TEM image and b) HRTEM image of ZnO thin film. Inset shows the corresponding SAED pattern.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4 Typical low and high magnification SEM images of the microneedle. a & b) Ag/AgCl microneedle. c & d) ZnO microneedle. Inset shows the general view of the microneedle.


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Page 27 of 31

100

(a)

150

pH 1.68

200 pH 4.01 250 pH 6.86 300 350 400 pH 9.18 450 500 0

200

400

600

800

Time (s) 100

(c)

100

Potential vs. Ag/AgCl (mV)

Potential vs. Ag/AgCl (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200

pH 2 pH 4

300

te Wa

400

r

pH 7

Sensor Stabilization (14 s)

500 0

pH 9

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (s)

Figure 5 a) Time dependence open circuit potential for various buffer solutions, b) pH sensing response with respect to fabricated microneedle and commercial Ag/AgCl electrode. c) continuous pH measurement with various buffer solutions



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6 Photo image of the in vivo experimental set-up a) mice on warming pad, b) mice brain after initial incision, c & d) testing CSF and bladder respectively.


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Page 29 of 31

348

280

352 354 356 358 ~ pH 7.45

360 362 364 366 368

(b)

285

Potential vs Ag/AgCl (mV)

Potential vs Ag/AgCl (mV)

Mice Cerebrospinal Fluid pH

(a)

350

Stablization Time (18 s)

370

Mice Bladder pH

290 295 300 305

~ pH 6.30

310 315 320

Stablization Time (30 s)

325 0

5

10

15

20

25

30

35

40

45

50

55

60

0

5

10

15

(c)

9

7.6

8

7.4

30

35

40

(d)

45

50

55

Estimated pH Measured pH

pH ~ 7.8 pH ~ 7.34

7

7.2

pH ~ 7.0 pH ~ 6.4

6

pH

7.0 6.8

5 4

6.6

3

6.4

2

6.2

1

6.0 5.8

25

10

8.0 7.8

20

Time (s)

Time (s)

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


M1 M2 M3 M4 M5

M1 M2 M3 M4 M5

Cerebrospinal Fluid

Bladder

0

Cerebrospinal Fluid

Bladder

Figure 7 pH measurements in a) mice cerebrospinal fluid, b) bladder pH. c) Repeatability of the fabricated microneedle with n-5 mice and d) comparison of estimated and measured pH via. conventional pH meter and microneedle respectively.


60


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 31

Table 1 A table of current and past in-vitro & in-vivo pH sensing studies presented.

Detection Method

Animal Type

Location

Type

Ref.

Luminescence

Human

Breast cancer tissue

In-vitro

7

Potentiometric

-

-

-

13

pH sensitive polymer membrane

Potentiometric

Dog

Arterial pH

In-vitro & In-vivo

Boron doped diamond microelectrode

Potentiometric

Mouse

Stomach

In-vivo

15

Flexible flat cable

Potentiometric

-

-

-

53

Human

Skin

In-vivo

54

55

Microneedle Type Optical fiber pH probe Iridium oxide on glass Wafers

Au serpentine mesh and gold-doped Potentiometric CVD graphene GrapheneModified Acupuncture Needle

Potentiometric

-

-

-

ZnO-Ag/AgCl on W microneedle

Potentiometric

Mice

Brain CSF & Bladder

In-vivo


52

Present Work

Page 31 of 31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Microneedle pH Sensor: Direct, Label-free, Real Time Detection of

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