An Automated Miniaturized Creatinine Sensing System

Son Vu Hoang Lai, David Gaddes, Srinivas Tadigadapa Department of Electrical Engineering

Material Research Institute, Penn State University University Park, Pennsylvania USA

Email: svl5279@psu.edu

Abstract — In this paper we report on the recently demonstrated, quartz resonator based thermal biosensor configured as a biomedical instrumentation system for continuous monitoring of kidney function based on the measurement of urinary creatinine excretion. The biosensor consists of a reaction chamber which is physically separated but located in close proximity to a micromachined quartz resonator-based temperature sensor. The highly effective coupling of heat from the reaction chamber to the quartz resonator due to close proximity (10 – 50 microns) and the extremely high absorption coefficient of quartz in the 8 – 12 μm wavelength range renders this system into a very sensitive thermal sensor design. The non-contact measurement results in no confounding mass loading effects on the quartz resonator and therefore provides clear calorimetric data. The sensor uses creatinine iminohydrolase enzyme immobilized on polystyrene films designed for disposable use. We have successfully integrated the sensor with a programmable miniaturized fluidic system from LabSmith® Inc. to form a miniaturized test system. In this paper we present the latest results on noncontact thermal sensor configuration integrated with miniature automated fluidic system that is capable of continuous monitoring of kidney function based on the measurement of urinary creatinine excretion.

INTRODUCTION Acute kidney injury (AKI) occurs in 5-7% of hospitalized

patients and results in a mortality rate of about 50%. Serum creatinine is the traditional standard measure of kidney function. However, it is insensitive to early changes in kidney function in an acute setting. In contrast, measurements of urine creatinine may be both sensitive and early indicators of acute kidney dysfunction. Techniques for continuous measurement of urine creatinine are currently unavailable and intermittent measurements based on techniques such as the Jaffe reaction are effort intensive and limit the practical frequency of measurements that can be performed. Until now the development of creatinine sensors has focused on

spectrophotometric techniques based on Jaffe reaction and electrochemical sensors [1, 2]. In spite of the success in demonstrating high sensitivity using these methods, electrochemical sensors, in addition to issues related to enzyme-stability, have reliability concerns due to fouling of the electrodes resulting in long-term drift and variations, while spectrophotometric methods are difficult to miniaturize for continuous, in-line creatinine measurements.

Calorimetry is a very powerful and an effective investigative tool for analyzing biochemical reactions [3] and offers a protected way to prevent drift in the response due to fouling of the base transducer. This endows thermal biosensors with unmatched operational stability for continuous monitoring, restricted only by the stability of the immobilized enzyme layer. The proposed Y-cut quartz resonator based sensor exhibits unmatched signal-to-noise performance and is ~250 times more sensitive than currently available temperature dependent resistors or thermocouples as demonstrated in our work on thermal sensors [4, 5]. This presents a unique opportunity to develop a potentially transformative, low cost, fast response, handheld sensing system for clinical diagnostic applications. In this paper we present the latest results on noncontact thermal sensor configuration [5, 6] integrated with miniaturized automated fluidic system that is capable of continuous monitoring of kidney function based on the measurement of urinary creatinine excretion.

PRINCIPLE OF OPERATION The operation of the sensor is based upon measurement of

the rise in temperature in real-time using the quartz crystal resonator (QCR) located in close proximity to the freestanding reaction chamber. Figure 1 shows the optical picture of the fluidic measurement set-up used for continuous flow enzymatic sensing. The miniaturized automated fluidic system is assembled from syringe pumps, reservoirs and 3-way valves capable of flowing a maximum of 100 µl of fluid per stroke that are commercially available from LabSmith® Inc and can be programmed using the LabSmith® controller module.

978-1-4673-4642-9/13/$31.00 ©2013 IEEE

Figure 2 show a schematic sequence of the automatic procedure for measurement. The automatic procedure for the measurement is set as follows: Syringe A takes the analyte from the analyte reservoir and then pumps the analyte onto the immobilized enzyme in fluidic channel located right above the sensor. The enzyme is immobilized on a polystyrene film which is clamped right above the quartz resonator sensor using an O-ring based compression seal. Thus, the enzyme catalyzed hydrolysis of the analyte occurs directly above and in close proximity to the thermal sensor (see Fig. 2(a)). After the calorimetric measurement is captured, air pump syringe is used to empty the reaction chamber of all the liquids in the reaction chamber via a blowout procedure (see Fig. 2(b)) and thereafter pumped to the waste. This is followed by a cleaning procedure in which PBS is pumped into the analyte chamber three times and the chamber is left in this state until the next cycle of measurement (see Fig. 2(c)). Prior to the next measurement the chamber is cleaned off the PBS by blowing air through the chamber before starting the analyte pumping.

Syringe A

Syringe B

Valve B

Valve A

Waste

Sensor with Immobilized Enzyme andFluidic Fixture

b

Reservoir

Figure 1: Optical picture of the packaged sensor with immobilized enzyme and fluid fixture integrated with a miniature automated fluidic system for urea & creatinine testing. Quartz resonators fabricated from crystal cuts such as Y-cut can be used as sensitive temperature sensors with unprecedented resolutions of up to 10-6

°C [7]. This phenomenological sensitivity of quartz crystals makes them a highly competitive alternative to the currently used uncooled thermal detectors such as vanadium oxide-based bolometers [8], thermopiles, and pyroelectric detectors [9]. Thus quartz crystal resonators can be configured as high performance thermal sensors and can achieve temperature resolutions as low as few mK. A. Y-Cut Quartz Resonator Biosensor Configuration

The resonance frequency of bulk acoustic wave quartz resonators is determined by the thickness of the resonator t as:

ρμ

tf

21

0 = (1)

where µ is the elastic modulus and ρ is the density of quartz. For quartz, µ = 2.95 x 1010

Pa and ρ = 2.65 x 103 kg/m3.

Analyte Pump

PBS Pump

Air Pump

3 port value

Reservoir

3 port connectorWaste

Sensor

atmosphere

Analyte Pump

PBS Pump

Air Pump

3 port value

Reservoir

3 port connectorWaste

Sensor

atmosphere

Analyte Pump

PBS Pump

Air Pump

3 port value

Reservoir

3 port connector

Sensor

Waste

atmosphere

a

c

b

Figure 2: Schematic illustration of the automatic pumping sequence during operation of the calorimetric sensor. (a) Pumping of 100 µl of analyte to initiate the measurement sequence, (b) Using air pump to clean the analyte chamber of the reacted analyte after the reaction, (c) Cleaning the chamber by pumping PBS and to keep the enzyme in PBS for optimal lifetime during operation.

Typical commercially available resonator crystals consist of 100 to 333 µm thick quartz with resonance frequencies in the 5 to 20 MHz range. Using bulk micromachining techniques, we have fabricated resonators from ~18 - 10 µm thick Y-cut quartz. These resonators have fundamental resonance frequencies of 80 - 167 MHz respectively. Y-cut quartz has a temperature sensitivity of +90 ppm/K yielding a temperature sensitivity of ~8 - 15 kHz/K.

EXPERIMENTAL RESULTS

B. Resonance Tracking and Calibration of Quart Resonator The resonance frequency of the fabricated micromachined

quartz resonators was monitored using an Agilent 4294A impedance analyzer. The impedance and phase were measured centered around the resonance frequency in a bandwidth of 500 kHz. Data was recorded for 801 frequency points within the bandwidth. Figure 3 shows the typical impedance curve obtained for the fabricated sensor. The temperature sensitivity of the resonator was measured around room temperature by placing the packaged resonator in an oven and allowing for the temperature to stabilize for 30 minutes before taking the resonance frequency measurement. Using extremely fast scan rates, each impedance scan takes around 3s. Since the typical biochemical reaction times are expected to be much faster, this method of tracking the resonance frequency is unsuitable for the current application. To overcome this limitation a new strategy as described in detail in our recent work has been used [10]. Briefly, the impedance analyzer is set to a fixed frequency at the mid-point between the two inflexion points in the impedance curve as shown in Fig. 3 and the change in impedance is monitored in real-time. For small temperature changes, an increase and/or decrease in the frequency, about the set quiescent frequency, translates into an increase or decrease in the value of |Z|. Using this method, continuous tracking of the resonance frequency reduces to real-time monitoring of impedance at a fixed frequency. A Labview® based program was developed to find the maximum slope factor in the linear region of impedance vs. frequency curve and to set-up the measurement frequency for real-time measurement of impedance change during the biochemical reaction at the set maximum slope point. With this new test method, the scanning time for every data point is only ~0.15 second, which resulted in 20 times more data points being recorded during experiments. C. Enzyme immobilization protocol

We have constructed a fluidic fixture made from Teflon and was sealed using a 250µm thick polystyrene film on which urease enzyme was immobilized using glutaraldehyde as cross-linker molecule. For the immobilization protocol, we used the method described by Mourzina et al [11]. Glutaraldehyde (25% aqueous solution), γ-aminopropyltriethoxysilane (98%), bovine serum albumin (BSA), urea, urease (jack bean urease

300 U/mg), monosodium and disodium phosphates were purchased from Sigma Aldrich. The buffer solutions were

Frequency (MHz)

Impe

danc

e (Ω

)

87.79 87.8 87.81 87.82 87.83 87.84 87.85 87.86 87.87 87.88 87.89 87.9-2000

-1500

-1000

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

Bias frequency

Real impedance RImaginary impedance X

Figure 3: Impedance of the micromachined quartz resonator spanning the resonance frequency shows a very high Q-factor of >10,000 for the 88 MHz devices prepared by mixing 67 mmol/L NaH2PO4 and 67 mmol/L Na2HPO4. Buffer pH values were adjusted by changing the volume ratio of Na2HPO4 and NaH2PO4 solutions. Buffer solutions of lower molarity were prepared by dilution with distilled water. A stock solution of urea (1 mol/L) was prepared in phosphate buffer solution, pH 6.11. Solutions with lower concentrations of urea were prepared by subsequent dilution with phosphate buffer. The pH was measured by means of a pH-meter. First, the polystyrene substrates were thoroughly cleaned and placed in a 10% aqueous solution of γ aminopropyltriethoxysilane. The pH of the solution was immediately adjusted with 6 mol/L HCl to pH 3.45. The polystyrene film was placed in this solution and heated to 75 °C in a water bath for about 3 h. The reaction can be schematically presented in the following way: ≡Si–OH + NH2(C3H6)Si(OR)3 ≡Si–O–Si(OR)2(C3H6)NH2

where R is an ethyl group. The derivatized substrates were washed with distilled water and heated to 100 °C overnight. Second, activation of the silanized carrier was performed via a reaction with glutaraldehyde. The reaction was carried out in a 2.5% solution of glutaraldehyde in 0.1 mol/L phosphate buffer at pH 7 at room temperature for about 3 h, followed by thoroughly washing out the excess glutaraldehyde with distilled water. The reaction can be schematically presented in the following way:

≡Si–O–Si(OR)2(C3H6)NH2 + CHO(CH2)3CHO ≡Si–O–Si(OR)2(C3H6)N=CH(CH2)3CHO

The resulting aldehyde carrier was used for covalent attachment of the proteins. Then 5 µl of a solution of urease (2250 U/ml) and BSA (60 mg/ml) in phosphate buffer, pH

7.0, was deposited onto the modified surface together with 3 µl of glutaraldehyde solution (2.5% in phosphate buffer, pH 7.0). The droplet was then thoroughly mixed and allowed to dry for 2 h. The sensor was stored in phosphate buffer at 5°C for further use. The same procedure was also prepared for creatinine stock solution and creatinine iminohydrolase enzyme immobilization. D. Urea and Creatinine Testing

Figure 4(a) shows the results of the tests for two concentration of urea at 23 °C temperature. Figure 4(b) shows the real-time output of the sensor for two concentrations of creatinine at 37 °C. The sensor is able to resolve 1 µM levels of creatinine which is an unprecedented level of sensitivity.

Time (s)

Imep

danc

e (Ω

)

0 20 40 60 80 100 120 140 160-50

0

50

100

150

200

250

3001M0.8M0M

Time (s)

Impe

danc

e (Ω

)

0 10 20 30 40 50-200

-100

0

100

200

300

400

500

600

700

800

900

1000100 mM100 μM

Figure 4: (a) Hydrolysis of Urea catalyzed by immobilized urease measured at 23 °C and (b) Real time sensor output for various concentrations of 100 μl of creatinine solution catalyzed by immobilized creatinine deiminase enzyme at 37 °C.

SUMMARY

In summary, the concept of a high-sensitivity, calorimetric, point-of-care device for automated bedside urine

testing based on a micromachined thermal sensor is presented. Immobilized urease and creatinine iminohydrolase enzyme on thin (250 µm) polystyrene films are used to measure the catalytic calorimetric output as a function of concentration in a flow set-up in an automated miniaturized fluidic system. The sensor design locates the active polystyrene film in close proximity to the quartz temperature sensor thereby providing an efficient heat coupling between the two. Measurements of various urea concentrations at room temperature and 37 °C and creatinine concentrations at 37 °C are presented. Concentrations of 50 mM for urea and 1 µM for creatinine have been measured. This paper presents the latest results on noncontact thermal sensor configuration integrated with miniature automated fluidic system that is capable of continuous monitoring of kidney function based on the measurement of urinary creatinine excretion.

ACKNOWLEDGMENT This work was supported by the National Science Foundation (NSF) under Grant ECCS 0925438. The use of facilities at the PSU Site of the NSF National Nanotechnology Infrastructure Network (NNIN) under Agreement 0335765 is acknowledged.

REFERENCES [1] A. J. Killard and M. R. Smyth, "Creatinine biosensors: principles and

designs," Trends in Biotechnology, vol. 18, pp. 433-437, 2000. [2] W. Joseph, "Electrochemical biosensors: Towards point-of-care cancer

diagnostics," Biosensors and Bioelectronics, vol. 21, pp. 1887-1892, 2006.

[3] B. Danielsson, "Calorimetric Biosensors," J. Biotechnology, vol. 15, pp. 187-200, 1990.

[4] Y. Zhang and S. Tadigadapa, "Calorimetric biosensors with integrated microfluidic channels," Biosensors and Bioelectronics, vol. 19, pp. 1733-1743, 2004/7/15 2004.

[5] K. Ren, P. Kao, M. B. Pisani, and S. Tadigadapa, "Monitoring biochemical reactions using Y-cut quartz thermal sensors," Analyst, vol. 136, pp. 2904-2911, 2011.

[6] S. V. Lai and S. Tadigadapa, "Calorimetric sensing system for real-time urea and creatinine measurements," in Sensors, 2012 IEEE, 2012, pp. 1-4.

[7] E. P. Eernisse, R. W. Ward, and R. B. Wiggins, "Survey of quartz bulk resonator sensor technologies," IEEE Transactions on Ultrasonics Ferroelectrics and Frequency Control, vol. 35, pp. 323-330, 1988.

[8] R. A. Wood, "Uncooled thermal imaging with monolithic silicon focal planes," presented at the Infrared Technology XIX, San Diego, CA, USA, 1993.

[9] A. Rogalski, "Infrared detectors: status and trends," Progress in Quantum Electronics, vol. 27, p. 59, 2003.

[10] M. B. Pisani, K. Ren, P. Kao, and S. Tadigadapa, Application of Micromachined Y-Cut-Quartz Bulk Acoustic Wave Resonator for Infrared Sensing, J Microelectromechanical Systems, vol. 20(1), pp. 288 -296, 2011.

[11] I. G. Mourzina, T. Yoshinobu, Y. E. Ermolenko, Y. G. Vlasov, M. J. Schöning, and H. Iwasaki, "Immobilization of Urease and Cholinesterase on the Surface of Semiconductor Transducer for the Development of Light- Addressable Potentiometric Sensors," Microchimica Acta, vol. 144, pp. 41-50, 2004.