Portable reconfigurable instrument for analytical determinations using disposable electrochemiluminescent screen-printed electrodes

Sensors and Actuators B 169 (2012) 46–53

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Portable reconfigurable instrument for analytical determinations using disposable electrochemiluminescent screen-printed electrodes M.A. Carvajal a , J. Ballesta-Claver b , D.P. Morales a , A.J. Palma a , M.C. Valencia-Mirón b , L.F. Capitán-Vallvey b,∗ a b

ECsens., Department of Electronics and Computer Technology, ETSIIT, Spain Department of Analytical Chemistry, Campus Fuentenueva, Faculty of Sciences, University of Granada, E-18071 Granada, Spain

a r t i c l e

i n f o

Article history: Received 13 November 2011 Received in revised form 19 January 2012 Accepted 31 January 2012 Available online 8 February 2012 Keywords: Electrochemiluminescence Reconfigurable electronics Portable instrument Screen-printed electrode Luminol and ruthenium chemistries

a b s t r a c t A portable reconfigurable instrument platform for electrochemiluminescence (ECL)-based disposable screen-printed electrodes is described. The reader unit consists of a potentiostat and a photodiode as a light-to-current converter integrated in the same instrument. For powerful and versatile analog conditioning, a very recent electronic solution has been included: a Field Programmable Analog Array (FPAA) capable of reconfiguring filter and gain stages in real time. To check the instrument’s performance as a sensor platform, two luminophores (tris(2,2 bipyridyl)ruthenium(II) and luminol) and two representative correactants widely used, triethylamine and cholesterol, were selected. The calibration functions obtained show a linear dependence with dynamic ranges from 0.05 to 10.0 mg l−1 for triethylamine, and 2.0·10−5 –1.4·10−4 M for cholesterol, with detection limits of 0.2 mg l−1 and 1.1·10−5 M, respectively, and a sensor-to-sensor reproducibility (relative standard deviation, RSD) of around 3.2% and 3.3% respectively at the medium-range level. This instrument offers a new advance in the portability of electrochemiluminescent determinations. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The market needs instrument miniaturization for fast analytical determinations in situ. Although different analytical techniques can be used, one technique of special interest for trace-analysis determinations is based on electrochemiluminescence (ECL). ECL is the light emitted when an electrochemical reaction occurs, and it offers advantages such as high selectivity and low detection limits. A number of instruments have been developed to measure the light from electroluminescent reactions. These processes are commonly controlled by an external potentiostat or an amperometric unit [1–3] that establishes potential differences or electric current flow between the cell electrodes, respectively. When there are no limitations for size or power supply, a good solution to collect a few photons generated by the ECL reaction is the use of a photomultiplier tube (PMT) [4], but these photodetectors need a very high voltage supply (up to thousands of volts). Blum et al. [5] studied a solution where fluidic micro-channels with two gold electrodes made of a printed circuit board (PCB) were used. This configuration was supplied by a PMT for collecting the ECL signal. The electrochemical reaction consists of hydrogen peroxide

∗ Corresponding author. E-mail address: [email protected] (L.F. Capitán-Vallvey). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2012.01.072

detection by reaction with electro-oxidized luminol. However, the bulky dimensions of PMTs make them inconvenient for portable instruments. Another technique for luminescence detection in the literature involves the use of CCD devices, such as CCD cameras and detectors [6]. However, in addition to complexity in their use and processing, the main disadvantage of these devices is that they need a working temperature of tens of degrees Celsius below zero to achieve good sensitivity. Organic and solid-state photodiodes as photodetectors to measure ECL radiation is also well described in the literature [7,8]. Photodiodes are small devices constantly undergoing improvements to their features as optical detectors, and are therefore easily integrated into a measurement system. The sensitivity of these photodetectors depends on the inverse polarization applied to them, which can vary from a few volts to hundreds of volts (in the case of avalanche photodiodes). Even in the photoconductive mode, these optoelectronic devices perform very well without supporting polarization circuitry. Therefore, photodiodes are well suited for portable instruments measuring luminescence from ECL reactions, which is the goal of this work. An example is the microanalysis system developed by Hosono et al., which consists of a reaction chamber with hydrophilic flow channels with gold valves as electrodes developed for aminoacid detection using Ru(bpy)3 2+ as the luminophore and a photodiode as detector [9]. In all of the cases mentioned above, the electrodes used were based on prefabricated gold or graphite small conductors.

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

However, the use of screen-printed electrodes (SPE) offers electrochemical measurements for portable instruments. An initial approach is to couple these devices to a commercial portable potentiostat, as Piermarini et al. report in developing biosensors for biogenic amines based on Prussian blue SPE cells [10]. However, at the moment, there is no good configuration for the use of these SPE cells for ECL measurements using commercial portable instruments. Our research group previously developed a hand-held luminometer for ECL lactate determination [11] based on conventional electronics. In the present work, a new reconfigurable electronic device is presented that improves the electronic platform. These devices, such as Field Programmable Analog Arrays (FPAA) for sensor signal conditioning, are powerful tools since they allow adapting the circuit’s parameter to the incoming signal requirements. This solution for sensor analog signal processing has been previously proposed in different fields ranging from biosignal acquisition [12] and environment smart sensor conditioning [13] to capacitive [14] and ultrasonic sensor conditioning [15], showing wide versatility because of its vast resources. An FPAA includes variable-gain amplifiers, a set of input and output terminals, and reconfigurable filters that allow fine bandwidth tuning, among other features. For the assayed analytes for this instrumentation, a tris(2,2 bipyridyl)ruthenium(II) (Ru(bpy)3 2+ ) was selected with triethylamine as the target molecule because the tertiary amine molecules are described in the field of DNA probe assays and other immunoassays having extensive applications, not only in clinical diagnostics, but also in forensic chemistry, environmental research, and pharmaceutical studies [16]. Another ECL method involved the enzymatic production of hydrogen peroxide by oxidases using luminol as the luminophore. In this case, we selected cholesterol as a characteristic example for the biological determinations. The inclusion, in a single instrument, of the photodiode, potentiostat, and reconfigurable electronics to adjust the analog processing to the ECL signal allow us to achieve the aim of portability. The main advantages of our design lie in that very portability, the low cost because of the use of a photodiode instead of a costly and bulky photomultiplier, versatility due to reconfiguration of the FPAA analog circuitry, and the possibility of using just a few microliters of sample with a batch system analysis.

47

reagents were obtained from Sigma (Sigma–Aldrich Química S.A., Madrid, Spain) except ethanol absolute, which was supplied by Panreac (Panreac Química SAU, Barcelona, Spain). As luminophores, we use a 10−2 M stock standard solution of luminol (5-amino-2,3dihydro-1,4-phthalazinedione) in saline phosphate buffer 0.2 M at pH 9.0 and 10−4 M of tris(2,2 -bipyridyl)dichlororuthenium(II) hexahydrate powder (Ru(bpy)3 2+ ) in 0.1 M of KNO3 salt, all from Sigma. The cholesterol stock solution was prepared with 0.19 g of cholesterol and 0.23 g of NaCl dissolved in 18.75 ml of ethanol, 6.25 ml of Triton® X-100, and water up to 25 ml. Reverse-osmosis water (Milli-Q Plus185 from Millipore, Molsheim, France) was used throughout. 2.3. Measurement process Volumes of 50 ␮l of different samples were placed on the SPE cell receptacle. For cholesterol, different standards were prepared from 2·10−2 M cholesterol stock solution in pH 9.0 phosphate buffer; for triethylamine determination, 10−4 M of TEA pH 8.5 Tris buffer 0.2 M was used. SPE cells were placed in a movable holder of the portable instrument covered with the lid holding the photodiode. Then, 10–12 pulses of 1.5 s were applied at 30 s intervals to measure the photodiode’s ECL emission. In the case of the cholesterol sample, a waiting time of 2 min was established for hydrogen peroxide enzyme production. 2.4. Apparatus and software

2. Experimental

The ECL emission from the screen-printed cells using the portable instrument was compared with a conventional instrument consisting of an H8529 photomultiplier (PMT) interfaced with a C8855 USB photo counting unit (both from Hamamatsu Technologies K.K., Shizuoka, Japan) connected to a computer. The potentiostat was an Autolab PGSTAT 128N (Metrohm Autolab B.V., Utrecht, The Netherlands) and a connector for the screen-printed electrodes supplied by Dropsens. The arrangement for ECL studies was described in previous works [17] and basically consists of a black box containing two black methacrylate holders that fit one inside the other, one to insert the electrochemical cell in a fixed position and the other to hold the PMT. A Crison digital pH-meter (Crison Instruments, Barcelona, Spain) with a combined glass-saturated calomel electrode was used to measure the pH.

2.1. Materials

3. Portable instrument description

The disposable sensor consists of a three-electrode screenprinted cell of graphite and gold, supplied by Dropsens (Oviedo, Spain). These cells comprise a working electrode, a counter electrode of the same material, and a silver pseudo-reference electrode on a 10 mm × 40 mm × 0.5 mm ceramic support. A receptacle for the solution in SPE cells was prepared by covering the electrode area with successive layers of white adhesive plastic tape up to 1 mm thick with an 8 mm diameter hole (ca. 50 ␮l volume) in the cell sensing area.

The system comprises a photodiode with an operational amplifier integrated in the same chip connected to the FPAA input for filtering and amplification, a potentiostat, and a microcontroller that controls all elements and digitizes the analog signal (Fig. 1). Microchip Technologies’ (USA) PIC18F2553 was chosen for this instrument.

2.2. Chemicals Saline buffers were prepared with phosphate 0.6 M at pH 6.0 for cholesterol oxidase (ChOx) enzyme preparation and pH 9.0 for cholesterol preparation using Na2 HPO4 and NaH2 PO4 , with NaCl 0.25 M as electrolyte. Tris Buffer (Trizma® Base) was used for triethylamine solution (TEA) (≥99.5%) with 0.2 M at pH 8.5. Other solutions, reagents, and solvents include ethanolic cholesterol stock solution of 2·10−2 M, ChOx from Streptomyces species (4.0 mg, 39.0 U mg−1 ), and Triton® X-100 (1.07 g ml−1 ). All of these

3.1. Instrument overview In order to avoid external illumination interference, the cell is placed inside a small dark drawer attached to the instrument housing. The photodiode’s output current needs to be converted into voltage by an I/V amplifier. This converter can be included in the photodiode case, on the same chip, or can be external. Before amplification, the output voltage of the I/V is connected to a 2 V level shifter, which is the FPAA reference voltage, with an adder stage consisting of an operational amplifier (TLC277, Texas Instruments, USA). The signal is filtered and amplified into the FPAA. The output signal is reduced to 2 V, the FPAA reference voltage, before the analog-to-digital conversion by the microcontroller. Finally, the

48

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

I0 (pA)

70 60

OPT301

50

S1227-66BR S9269

40

S9270

30

S1227-1010BR

20 10 0 0

20

40

60

80

100

ILED (nA) Fig. 2. Output current of the studied photodiodes.

3.2. Photodiode selection

Fig. 1. (A) Luminometer block diagram; (B) picture of ECL instrument with the disposable sensor connection.

results are stored in a 1 Mbit EEPROM memory. The output is digitized by the microcontroller with the internal 12-bits analog to digital converter. The results are stored in the EEPROM memory and downloaded to the computer at the end of the experiment. The M24C1024 (Microchip, USA) memory communicates with the microcontroller via I2C, reducing the number of wires and permitting the connection of new devices. The potentiostat is the electronics in charge of starting the ECL reaction by applying voltage pulses. Basically, it consists of a digital-to-analog converter (DAC) and analog circuitry to amplify and shift the voltage output. In Section 3.2, the pulse configuration is described in detail. Finally, the luminometer can be controlled by a computer via USB. An ad hoc software application has been developed to allow remote configuration and downloading of results. The instrument can be powered by USB or by a single battery of over 6 V, for example a PP3 9 V battery. However, the potentiostat must be able to produce bipolar pulses, and therefore a negative voltage source is needed. As a result, a switch power supply based on a DC/DC converter has been included. We used the MAX743 (Maxim-Semiconductor, USA), which is a DC/DC up-converter and inverter that provides a bipolar power supply voltage of ±15 V or ±12 V, with a typical power efficiency of 82%. The MAX743 has been configured to produce ±12 V and to reduce power-switching noise; two linear regulators have been included to reduce the voltage to ±5 V, making it as noise free as possible. This bipolar voltage source biases the level shift stages at the input and output of the FPAA and the potentiostat. The DC shift is required because the FPAA voltage reference is 2 V instead of 0 V like the rest of the analog circuit. Instrument power consumption is around 210 mA, of which 110 mA is consumed by the FPAA. In fact, the power consumption of the FPAA is the most important drawback to its use in portable instrumentation. However, the requirements of our instrument can be satisfied by a single 4.5 V battery, four AAA batteries, or a USB 2.0 port.

We studied two photodiodes without an integrated I/V converter: S1227-66BR and S1227-1010R (Hamamatsu, Japan), with an active area of 33 and 100 mm2 , respectively. To achieve a high enough gain factor, a T net resistor was used as the feedback converter resulting in an effective resistance of 5.2 G. Another three photodiodes with a built-in I/V amplifier were tested: the S9269 and S9270 (Hamamatsu, Japan), with an active area of 33 and 100 mm2 respectively, and the OPT301 (Texas Instruments, USA), with an area of 5.2 mm2 . The S9269 and the S9270 have an internal resistance of 1 G and the OPT301 an internal resistance of 1 M. In the last one, an additional external T net resistance was added to reach a value of 5.2 G. The output currents of the various photodiodes are plotted in Fig. 2. As expected, the most sensitive photodiodes were the S9270 and the S1227-1010BR due to their larger sensitive area. We selected the S9270 photodiode since it also includes a built-in I/V converter, which gives it better interference immunity. 3.3. Potentiostat This instrument’s potentiostat is based on a previous design [11] with some modifications. This potentiostat (Fig. 3) consists of some operational amplifiers, a digital-to-analog converter (DAC), and three outputs: reference electrode (RE), working electrode (WE), and the counter electrode (CE). The initial voltage pulse is produced by a DAC of 16 bits, the DAC8571 (Texas Instruments, USA), which communicates with the microcontroller via an I2C bus. This pulse is amplified and shifted with a DC level to provide different voltages between the working and reference electrodes. It must be taken into account that no current must flow through the RE, but nevertheless electrons have to be collected or provided in redox reactions. Therefore, a third electrode (CE) is included, which

I2Cbus

RF

DAC VREF

RSC CE

RE WE

Fig. 3. Potentiostat electronic scheme.

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

49

Fig. 4. Output voltages of the potentiostat applying a monopolar pulse: RE in blue, WE in red, and CE in green. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

permits the injection or drains the current from the WE. The WE is virtually grounded and connected to an I/V converter to measure current during the reaction. The operational amplifier used in this design is the TLC277 (Texas Instruments, USA) due its very high input impedance and low power consumption. In a previous design, the CE, which was the output of one of the operational amplifiers, could be saturated if the solution under test was not deposited on the electrode. When the solution is deposited, negative feedback occurs and the voltage between the WE and RE can be controlled by the DAC. The main problem of this configuration is that a very short time pulse of around 5 V might be applied to the test solution, starting the reaction early or damaging the reagent. This problem can be solved using a micro-relay that makes feedback possible and commutes the pulse voltage. Nevertheless, power consumption increases and relay reboots can produce spurious pulses. Therefore, we have included a resistor (RF ) between the CE and RE to make negative feedback possible and prevent a spurious pulse. This resistor must be as high as possible to reduce current flow through the RE. We used a resistor of 10 M, which is much higher than the resistance value of the solutions studied in this work (around 300 k). In addition, a reference voltage is included for analog-to-digital conversion and for stabilizing the potentiostat pulses. In this work, only mono-polar pulses in chronoamperometric form for cholesterol and TEA ECL measurements were performed. However, the possibility of providing bipolar pulses has been included for developing a more flexible instrument that can be used in future to launch other chemical reactions. In Fig. 4, a voltage waveform is plotted with the three-electrode cell. The instrument can configure the applied pulses by varying the maximum voltage value (positive and negative) and the rise and fall times. 3.4. FPAA configuration The design of analog circuits for sensor signal processing is a crucial stage in electronic instrumentation that deals with changing features in the incoming signal, such as an amplitude drop and/or noise increase. When static circuits are designed, they are built with the restriction that they must work in the worst-case scenario. This generally translates to a reduction in system resolution. On the other hand, if the designed circuit adapts its parameter to the specific incoming signal, it results in higher resolution and a better S/N ratio. This circumstance can be achieved using an FPAA device that

Fig. 5. Graphic interface from the software tool where the FPAA-hosted conditioning circuitry has been implemented.

accommodates the analog conditioning circuitry allowing multiple circuit implementations within the same hardware device. For this instrument, an AN221E04 FPAA device from Anadigm® was chosen. This device is based on switched-capacitor technology, which allows a wide set of analog functions named Configurable Analog Modules (CAM) to be implemented. The device’s digital interface lets us perform dynamic reconfiguration from a microcontroller host or auto-configuration from an EPROM. The device configurations are built with a software interface that creates the configuration file to be loaded in the device configuration RAM. This RAM performs all the connections and clocks frequency selection in the device’s internal circuitry to produce the analog conditioning path. Since the input signal from the I/V converter integrated with the photodiode presents different maximum amplitudes depending on the solution used, the circuit should change its total gain to cope with it. In addition, the photodiode signal could suffer from electromagnetic noise from surrounding power lines. Finally, highfrequency noise is added from the environment and electronic lines and devices that must be minimized. Therefore, Fig. 5 depicts the FPAA-implemented hardware, which consists of three main CAMs and additional input and output cells configuration. Table 1 summarizes the main features of these CAMs. Following the signal path in Fig. 5, the first processing module is a low-pass filter at the input cell configured for single-ended input signals that must be referenced to analog common (2 V) or a spawn range within 0–4 V. The anti-alias filter is implemented with two cascaded, single-pole, continuous-time filters. The next module is a notch filter implemented in a biquadratic (two pole) structure whose aim is to eliminate the incoming power line interferences, and so its corner frequency is set to 50 Hz. The subsequent element in the signal path is a low-pass biquadratic filter with a corner frequency set to 20 Hz and a gain set to 10. This filter eliminates high-frequency noise that can affect the input signal. These two filter modules may change their corner frequencies and gains by a device reconfiguration from the host microcontroller in response to a change in input signal quality. The last module is designed to eliminate the internal offset generated within the device by means of an inverting summing stage that allows us to select independent gains to each input. Thus, the −3 V source offers the required

50

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

Table 1 CAMs employed for signal conditioning. Name

Description

Parameters

Filter clock

InputCell1 OutputCell2 SumInv

Differential input with low pass anti-alias filter Differential voltage output with low pass anti-alias filter Adding inverting amplifier with selectable independent input gain

– – 5.229 kHz

FilterBiquad

Biquadratic (two pole) filter set to Band Stop topology

FilterBiquad

Biquadratic filter set to low pass topology. Sampling phase 1

Filter corner frequency 76 kHz Filter corner frequency 76 kHz Gain 1 (UpperInput): 1 Gain 2 (LowerInput): 0.2 Band stop frequency 50 Hz Pass bands gains: 1 (DC), 1 (HP); Quality factor 30 Filter corner frequency 20 Hz Flat band gain: 10 Quality factor: 15

Voltage DC

Polarity: negative (−3 V)

Light Intensity (a.u.)

60

the beginning of the pulse, a narrow peak appears due to potentiostat switching. This peak is a typical response to switching power sources, and it is due to the slow response of this source type, happening even without solution on the electrode. Therefore, this peak cannot be taken into account for ECL measurements. Therefore, a smoothing filter has been programmed into the microcontroller to eliminate the spurious peak. The microcontroller waits for 225 ms to pass from the onset of the potentiostat pulse and then seeks the light maximum, ignoring peaks prior to it. The response of the light peaks is relatively slow due to the FPAA analog filtering, thus delaying the response. This fact is not a problem because the pulse is wide enough and the maximum approaches the correct value, as Fig. 6 shows. The microcontroller finds the maximum value and subtracts the baseline (46 approx. in Fig. 6). This signal processing was used in two chemical systems:

Switching peak End of digital filtering

50

45 Potentiostat pulse

40 136

137

138

139

5.229 kHz

–

Light intensity maximum

55

5.229 kHz

140

141

142

143

t (s) Fig. 6. Tenth pulse of cholesterol determination for 2·10−5 M.

(A) Ru(bpy)3 2+ –TEA system offset compensation. Finally, the output cell hosts a low-pass filter that eliminates internal noise from the switched capacitances. All the internal CAMs in the signal path are clocked at the same rate to prevent clock mismatch from one to another that might disturb the signal.

In this case, the signal is obtained working in the chronoamperometric mode generating 10 pulses. Regarding the Ru(bpy)3 2+ concentration used, we studied different concentrations from 10−5 M to 10−2 M, finding that 10−4 M offers the best results for TEA determination. As described in the bibliography [18], the optimum excitation voltage corresponds to 1.3 V with a Ag/AgCl as reference electrode, an aspect that we checked using the graphite SPE cells (Ag pseudo-reference), obtaining the same value. Then, the measurement conditions were: (a) applied potential (1.3 V); (b) time between pulses, with 30 s being better for sensitivity; (c) pulse time of 1.5 s. The analytical signal was the average of the last four pulses due to the electrochemical decay effect in the first pulses. The blank signal is owing to the well-known interaction of the Ru(bpy)3 3+ and hydroxide ions formed from the basic media [19], obtaining then a decreasing emission signal when consecutive 1.3 V excitation peaks are applied. Fig. 7 shows the maximum filtered emission peaks of the blank and the standard.

4. Experimental results 4.1. Measurement conditions The instrument is equipped with the electrochemical tools for the different types of ECL analytical signals. The pulse intensity of the collected light shows a direct relationship with sample concentration. However, several pulses of the same concentration must be studied over time to see the peak profiles to discriminate and eliminate the intensity of blank signals. Fig. 6 shows a typical pulse of light obtained by our instrument during the potentiostat’s voltage pulse. As can be seen, at

24

A

Filtered light intensity (a.u.)

Filtered light intensity (a.u.)

20 TEA 2 ppm

16 12 8

Blank signal

4 0 0

2

4

6

Pulse nº

8

10

12

B

20 Cholesterol (4.5·10-5 M)

16 12 8

Blank signal

4 0

0

2

4

6

8

10

12

14

Pulse nº

Fig. 7. Filtered ECL emissions of the maximum peaks obtained. Different pulses were applied for: (A) TEA system (1.3 V); (B) cholesterol system (0.5 V).

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

(B) Luminol–Cholesterol system

51

800

600 500 400 300 200 100

Gain = 20

Light (a.u.)

Light Intensity (a.u.)

700

Different parameters were studied for the chemical conditions of the required reagents for cholesterol determination: (a) concentration of luminol: 1 mM. A higher value decreases the signal due to the accumulation of products or the dismutation of reactive intermediates rather than reagent depletion [20,21]; (b) if oxygen is present, the ChOx enzyme can catalyze the oxidation of cholesterol to produce H2 O2 and 5-cholesten-3-one [22]. This H2 O2 can react with electro-oxidized luminol to yield an ECL emission. The optimum concentrations were 520.0 IU ml−1 ; (c) The optimum pH for ChOx is 7.0 [23], whereas the luminol reactivity with H2 O2 increases with medium alkalinity (over 8.0) due to the contribution of hydroxyl groups to catalyze the ECL reaction [24]. The 9.0 maximum was chosen as a compromise pH value considering both effects. The blank signal obtained is due to the reaction of the dissolved oxygen in solution with the oxidized luminol and we do not observe any contribution due to ChOx. Fig. 7 shows the profile when consecutive pulses were given. The conditions for ECL cholesterol measurement were twelve 0.5 V pulses for 1.5 s, with a rest time of 30 s between two consecutive pulses. In our case, the best analytical parameter corresponds to the average maximum of the last six pulses, which gives the best repeatability performance. After studying the sample volume in the screen-printed electrode, 50 ␮l was chosen as the optimum amount. In this case, the elapsed time between sample addition and the ECL measurement was very important because the longer the waiting time, the more H2 O2 production and hence more ECL output. Therefore, 2 min from sample addition was selected. As mentioned above, the average of the maxima regarding the baseline (last 4 pulses for TEA and 6 for cholesterol) is our analytical signal. The standard deviation and instrumental error have been considered for the uncertainty calculation. The results are reported in the subsection below.

4000 2000 0 0

t (s)

200

0 0

2

4

6

8

10

-1

[TEA] (mg l )

50 40 30 20 10

Gain = 10

light (a.u.)

Light Intensity (a.u.)

60

700 600 500 400 0

0 0.0E+00

5.0E-05

1.0E-04

100

t (s)

1.5E-04

[Cholesterol] (M) Fig. 8. Calibration curves of TEA and cholesterol (experimental data of a standard included in the inset).

in the case of the TEA determination. Linear calibrations were obtained as shown in Fig. 8 and Table 2. Table 2 shows a comparative study using the portable instrument presented in this work versus the conventional instrumentation described above. The portable instrument offers a wide dynamic linear range and better linear correlation, providing a better fit of the experimental data and improved sensitivity. The detection limit is in consonance with the intercept of the linear curve because the lower the intercept value (low noise level), the greater the accuracy (low RSD blank values). However, the sensitivity of the conventional method is superior to the

4.2. Analytical parameters For the evaluation of this portable luminometer, two calibrations were performed with the two chemicals involved in this work. Different cholesterol concentrations from 1.0·10−5 to 8.0·10−4 M were tested and concentrations from 0.05 to 10 mg l−1 were used

Table 2 Analytical characteristics for portable and conventional instrumentation. Parameter

Portable

Conventional −1

TEA (mg l Linear range Intercept (a.u.) Slope R2 Detection limit RSD blank (%) RSD sampleb (%) a b

)

Cholesterol (M) −5

0.05–10.0 17.6 63.2 0.998 0.2 1.9 3.2

−4

2.0·10 –1.4·10 2.04 3.1·106 0.987 1.1·10−5 23.2a 3.3

TEA (mg l−1 )

Cholesterol (M)

1.2–10.0 39.32 34.12 0.985 0.37 10.3 7.2

2.5·10−5 –1.0·10−4 106.3 4.77·106 0.945 2.3·10−5 2.5 7.8

A lower blank signal generates high RSD. In the middle of the linear range (2 mg l−1 for TEA, and 6.0·10−5 M for cholesterol).

Table 3 Analytical characteristics of ECL procedures for cholesterol and TEA from literature. Analyte Cholesterol (M) Cholesterol (M) TEA (mg l−1 ) TEA (mg l−1 ) a

Capillary electrophoresis.

Linear range −6

Detection limit −4

1.0·10 –4.0·10 2.4·10−6 –3.0·10−4 0.001–50 50–5060

−7

8.0·10 2.4·10−6 8.0·10−4 –

RSD sample (%)

Remarks

Reference

2.5 4.8 2.8 –

PMT/HPLC PMT/sensor PMT/CEa PMT/biosensor

[25] [26] [27] [28]

52

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53

portable instrument, as the calibration slopes show. In contrast, the portable system offers improved repeatability over the conventional method. In a comparison with different methods in the bibliography, the analytical parameters in Table 3 show that the linear range is wider using separation techniques and the detection limit is lower for our portable device, with the best results obtained when the separation techniques such as HPLC or CE were coupled to a PMT and a potentiostat. However, the repeatability values are similar to using conventional methods, indicating that our method is quite accurate.

5. Conclusions A novel portable instrument based on a photodiode and a simple potentiostat is presented as suitable for ECL measurements. The main advantage of this device compared with previous designs is the inclusion of a reconfigurable circuit for analog conditioning. This electronic device can measure different ECL reactions to obtain the concentrations of trace compounds using the same equipment. Although the switched capacitor technology (FPAA core) might have been expected to produce electric interference problems, it has performed well even with these low-level signals. The instrument versatility allows working with a range of procedures such as Ru(bpy)3 2+ luminophore for TEA measurements and luminol chemistry for clinical applications exemplified by a cholesterol determination. Although the ECL determination has been measured in batch mode, it is possible to use it in a continuous system by adapting a suitable flow cell. In principle it might seem that a photodiode-based system for collecting light would offer a less sensitive electrochemiluminescent determination. However, in this work we have proved that this methodology offers low noise limits that make up for any possible drawbacks that in principle it might have compared to conventional instruments. Therefore, this device offers an inexpensive and extremely versatile means of producing more accurate measurements in the electrochemiluminescent biosensors field.

Acknowledgements This work has been funded by the Junta de Andalucía, Spain (Projects P09-FQM-5341, P08-FQM-3535), projects that were partially supported by European Regional Development Funds (ERDF).

[8] O. Hofmann, P. Miller, P. Sullivan, T.S. Jones, J.C. de Mello, D.D.C. Bradley, A.J. de Mello, Thin-film organic photodiodes as integrated detectors for microscale chemiluminescence assays, Sens. Actuators B 106 (2005) 878. [9] H. Hosono, W. Satoh, J. Fukuda, H. Suzuki, Microanalysis system based on electrochemiluminescence detection, Sens. Mater. 19 (2007) 191. [10] S. Piermarini, G. Volpe, R. Federico, D. Moscone, G. Palleschi, Detection of biogenic amines in human saliva using a screen-printed biosensor, Anal. Lett. 43 (2010) 1310. [11] A. Martinez-Olmos, J. Ballesta-Claver, A.J. Palma, M.D.C. Valencia-Miron, L.F. Capitan-Vallvey, A portable luminometer with a disposable electrochemiluminescent biosensor for lactate determination, Sensors 9 (2009) 7694. [12] D.P. Morales, A. Garcia, E. Castillo, M.A. Carvajal, J. Banqueri, A.J. Palma, Flexible ECG acquisition system based on analog and digital reconfigurable devices, Sens. Actuators A 165 (2011) 261. [13] D.P. Morales, A. Garcia, A. Martinez-Olmos, J. Banqueri, A.J. Palma, Digital and analog reconfiguration techniques for rapid smart sensor system prototyping, Sens. Lett. 7 (2009) 1113. [14] S. Callegari, G. Merendino, A. Golfarelli, M. Zagnoni, M. Tartagni, Applicability of field programmable analog arrays to capacitive sensing in the sub-pF range, Analog Integr. Circuits Signal Process. 47 (2006) 39. [15] A. Baccigalupi, A. Liccardo, Field programmable analog arrays for conditioning ultrasonic sensors, IEEE Sens. J. 7 (2007) 1176. [16] P. Pastore, D. Badocco, F. Zanon, Influence of nature, concentration and pH of buffer acid–base system on rate determining step of the electrochemiluminescence of Ru(bpy)32+ with tertiary aliphatic amines, Electrochim. Acta 51 (2006) 5394. [17] J. Ballesta-Claver, M.C. Valencia-Miron, L.F. Capitan-Vallvey, Disposable electrochemiluminescent biosensor for lactate determination in saliva, Analyst 134 (2009) 1423. [18] M.M. Richter, Electrochemiluminescence (ECL), Chem. Rev. 104 (2004) 3003. [19] R.D. Gerardi, N.W. Barnett, S.W. Lewis, Analytical applications of tris(2,2 bipyridyl)ruthenium(III) as a chemiluminescent reagent, Anal. Chim. Acta 378 (1999) 1. [20] Y. Huang, W. Liu, Flow-injection chemiluminescence determination of phentolamine based on its enhancing effect on the luminol–potassium ferricyanide system, J. Pharm. Biomed. Anal. 38 (2005) 537. [21] B.B. Kim, V.V. Pisarev, A.M. Egorov, A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays, Anal. Biochem. 199 (1991) 1. [22] J. MacLachlan, A.T.L. Wotherspoon, R.O. Ansell, C.J.W. Brooks, Cholesterol oxidase: sources, physical properties and analytical applications, J. Steroid Biochem. Mol. Biol. 72 (2000) 169. [23] W. Richmond, Preparation and properties of a cholesterol oxidase from Nocardia sp. and its application to the enzymatic assay of total cholesterol in serum, Clin. Chem. 19 (1973) 1350. [24] H. Chu, W. Guo, J. Di, Y. Wu, Y. Tu, Study on sensitization from reactive oxygen species for electrochemiluminescence of luminol in neutral medium, Electroanalysis 21 (2009) 1630. [25] Q. Zhu, M. Han, H. Wang, L. Liu, J. Bao, Z. Dai, J. Shen, Electrogenerated chemiluminescence from CdS hollow spheres composited with carbon nanofiber and its sensing application, Analyst 135 (2010) 2579. [26] C.A. Marquette, S. Ravaud, L.J. Blum, Luminol electrochemiluminescence-based biosensor for total cholesterol determination in natural samples, Anal. Lett. 33 (2000) 1779. [27] B. Yuan, J. Huang, J. Sun, T. You, A novel technique for NACE coupled with simultaneous electrochemiluminescence and electrochemical detection for fast analysis of tertiary amines, Electrophoresis 30 (2009) 479. [28] E.S. Jin, B.J. Norris, P. Pantano, An electrogenerated chemiluminescence imaging fiber electrode chemical sensor for NADH, Electroanalysis 13 (2001) 1287.

Biographies References [1] J. Gautron, J.P. Dalbera, P. Lemasson, Electroluminescence of the electrolyte/nZnSe junction under anodic polarization, Surf. Sci. 99 (1980) 300. [2] J.C. Fidler, W.R. Penrose, J.P. Bobis, A potentiostat based on a voltage-controlled current source for use with amperometric gas sensors, IEEE Trans. Instrum. Meas. 41 (1992) 308. [3] X. Zheng, Z. Zhang, B. Li, Flow injection chemiluminescence determination of captopril with in situ electrogenerated Mn3+ as the oxidant, Electroanalysis 13 (2001) 1046. [4] H. Zhou, Z. Zhang, D. He, Y. Hu, Y. Huang, D. Chen, Flow chemiluminescence sensor for determination of clenbuterol based on molecularly imprinted polymer, Anal. Chim. Acta 523 (2004) 237. [5] P. Pittet, G.N. Lu, J.M. Galvan, R. Ferrigno, L.J. Blum, B.D. Leca-Bouvier, PCB technology-based electrochemiluminescence microfluidic device for low-cost portable analytical systems, IEEE Sens. J. 8 (2008) 565. [6] N. Momeni, K. Ramanathan, P.O. Larsson, B. Danielsson, S. Bengmark, M. Khayyami, CCD-camera based capillary chemiluminescent detection of retinol binding protein, Anal. Chim. Acta 387 (1999) 21. [7] A. Hemmi, K. Yagiuda, N. Funazaki, S. Ito, Y. Asano, T. Imato, K. Hayashi, I. Karube, Development of a chemiluminescence detector with photodiode detection for flow-injection analysis and its application to l-lactate analysis, Anal. Chim. Acta 316 (1995) 323.

Miguel A. Carvajal Rodríguez was born in 1977 in Granada (Spain). He received the M.Sc. degree in Physics in 2000 and the M.Sc. degree in Electronic Engineering in 2002, both from the University of Granada; and the Ph.D. degree in Electronic Engineering from the University of Granada in 2007. Currently he works as an Assistant Professor at the University of Granada. His research interests include the effects of irradiation and post-irradiation in MOSFET transistors, gas sensor and their application to handheld instrumentation. Julio Ballesta-Claver was born in 1977 in Granada (Spain). He received the MSc degree in Chemistry (2000) and the PhD degree in Analytical Chemistry (2009), both from the University of Granada. He is currently working as Researcher at the ECsens group, Department of Analytical Chemistry, Granada. His current research interests include electrochemiluminescence, chemical sensors and biosensors, electroanalytical techniques and polymer sciences. Diego P. Morales received the MASc degree in Electronic Engineering and the PhD degree in Electronic Engineering from the University of Granada (Granada, Spain) in 2001 and 2011, respectively. He was an Associate Professor at the Department of Computer Architecture and Electronics, Universidad de Almería (Almería, Spain) before joining the Department of Electronics and Computer Technology at the University of Granada, where he currently serves as an Assistant Professor. His current research interests include the combination of digital and analog programmable technologies for smart instrumentation and FPGA-based signal processing systems.

M.A. Carvajal et al. / Sensors and Actuators B 169 (2012) 46–53 Alberto J. Palma López was born in 1968 in Granada (Spain). He received the BS and MSc degrees in Physics (Electronics) in 1991 and PhD in Physics (1995) from the University of Granada, Spain. He is currently Full Professor at the University of Granada. Since 1992, he has been working on trapping of carriers in different electronic devices (diodes and MOS transistors) including characterisation and simulation of capture cross sections, random telegraph noise, and generationrecombination noise in devices. From 2000, his current research interest is the study of the application of MOS devices as radiation sensors and the electronic instrumentation design directed to portable, low cost electronic systems in the fields of chemical and physical sensors. He is co-author of about 100 peer-reviewed scientific papers and congress communications and 5 patents. María del Carmen Valencia-Mirón, PhD in Chemistry, currently serves as Associate Professor at the Department of Analytical Chemistry in the University of Granada

53

(Spain). Her current research interests include the study of chemiluminescence and electrochemiluminescence, disposable sensors and flow analysis. Luis Fermín Capitán-Vallvey, Full Professor of Analytical Chemistry at the University of Granada, received his BSc in Chemistry (1973) and PhD in Chemistry (1986) from the Faculty of Sciences, University of Granada (Spain). In 1983, he founded the Solid Phase Spectrometry group (GSB) and in 2000, together with Prof. Palma López, the interdisciplinary group ECsens, which includes Chemists, Physicists and Electrical and Computer Engineers at the University of Granada. His current research interests are the design, development and fabrication of sensors and portable instrumentation for environmental, health and food analysis and monitoring.