Sensors and Actuators B 155 (2011) 206–213

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Bioelectronic system for the control and readout of enzyme logic gates
Joshua Ray Windmiller a , Padmanabhan Santhosh a , Evgeny Katz b,∗ , Joseph Wang a,∗∗
a
Department of NanoEngineering, University of California – San Diego, La Jolla, CA 92093, USA
b
Department of Chemistry and Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699-5810, USA

a r t i c l e i n f o a b s t r a c t

Article history: In this work we describe the development of a novel microelectronic backbone configured specifically
Received 15 October 2010 for the control of biocomputing systems applied to diagnostic merits. The operation of the sensor sys-
Received in revised form tem is validated towards the rapid assessment of pathological conditions arising from soft tissue injury
19 November 2010
(STI) and abdominal trauma (ABT) using NAND and AND Boolean enzyme logic gates, respectively. The
Accepted 22 November 2010
miniaturized 19 × 19 mm device employs a custom-designed three-electrode potentiostat coupled with
Available online 27 November 2010
an integrator, voltage amplifier, comparator, and digital logic and is easily interfaced with a screen-
printed electrode contingent. By implementing an adjustable threshold comparator, a precise decision
Keywords:
Enzyme logic
threshold could be established corresponding to pathological levels of the target biomarkers. As a result,
Potentiostat a rapid amperometric analysis tendered the diagnosis in a straightforward ‘YES’/‘NO’ digital format via
Sensor the illumination of a light emitting diode. Using low quiescent current voltage regulators, the device is
Screen-printed electrode able to achieve microwatt power operation and can be sustained by a single 3 V coin-cell battery for over
Chronoamperometry 45 h under continuous use. The low-power, low-cost, and miniaturized device meets the requirements of
field-deployable logic gate amperometric sensors. Such a reconfigurable micro-/bioelectronic logic-based
multi-parameter sensing system shows considerable potential for the assessment of key analytes in a
multitude of relevant clinical, security, and environmental applications where go/no-go readout, rapid
measurement, device miniaturization, and extended longevity on battery power are key requirements.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction functions. However, the interface between the electronic and

chemical constituents of biosensors remains as one of the key limi-
Advanced functional biosensors have attracted a significant tations towards the realization of miniaturized, low power devices.
research following over the past several decades due to the prospect Most notably, parallel detection must be employed when multi-
of improving the healthcare and quality of life for those who inte- variate chemical analysis is required. In such schemes, a specific
grate such diagnostic measures into their daily routine [1]. In a sensing element is required to monitor the level of each unique
number of utilitarian point-of-care applications, the presentation of chemical entity [3]. Consequently, highly parallel sensor arrays
a pathological assessment in simple ‘YES’/‘NO’ terms rather than in necessitate the utilization of multiple electronic sensing elements
quantitative form may be of greater pertinence to the non-technical for controlling multiple working electrodes, thereby scaling power
operator. As such, a biosensor device that enables straightforward consumption and device size accordingly. Alternatively, chemical
and rapid readout with minimal operator intervention would be reactions may be engineered to manipulate multiple chemical enti-
well-positioned for end-user needs in the healthcare domain. ties in the chemical domain prior to transduction to the electronic
Microelectronics have traditionally been leveraged to achieve domain for further processing. In this manner, chemical signal pro-
the miniaturization that is a core requirement of modern electro- cessing can be exploited in order to enable the detection of several
chemical biosensors as well as to extend their operating times on analytes with a single sensing contingent. This would enable a fur-
battery power [2]. Advances in microelectronics have resulted in ther degree of miniaturization and reduced power consumption
major changes to electroanalytical instrumentation, with minia- while maintaining the overall functional ability of the complex
turized and inexpensive integrated circuits performing many system.
Recent developments in the area of biochemical computing and
biomolecular logic systems have resulted in the demonstration of
enzyme-based logic gates that resemble conventional electronic
∗ Corresponding author. Tel.: +1 315 268 4421; fax: +1 315 268 6610. Boolean logic gates [4]. These enzyme-based logic gates are able
∗∗ Corresponding author at: Department of NanoEngineering, University of Cali-
to integrate two or more physiologically relevant inputs and pro-
fornia, Mail Box 0448, La Jolla, CA 92093, USA. Tel.: +1 858 246 0128;
fax: +1 858 534 9553.
cess the biochemical information biocatalytically to yield a single
E-mail addresses: [email protected] (E. Katz), [email protected] (J. Wang). output [5]. Such enzyme logic gates have been shown to enable

0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2010.11.048
 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213 207

a high-fidelity assessment of pathophysiological status in unam- A Gly-Gly buffer solution was prepared at 50 mM concentra-
biguous ‘YES’/‘NO’ terms corresponding to the presence of one or tion with 6.7 mM MgAc to provide the magnesium ion activator for
more injuries [6–11]. Advantageously, the enzyme logic paradigm CK. The buffer was then titrated with 1 M KOH to create a solution
facilitates straightforward chemical analysis since the output of with pH value of 7.95 (while providing the potassium ion cofac-
these gates is truly binary and unambiguous in nature. Accord- tor essential for PK). All reagents employed in the soft tissue injury
ingly, multiple-input enzyme logic biosensors call for a redesign (STI) gate were prepared in this buffer solution. MG was employed
of the supporting electronics due to the substantially dissimilar as a mediator to enable the low-potential oxidation of NADH.
operational principles embodied by such logic gates when com- A potassium phosphate buffer solution was prepared at 50 mM
pared with conventional single-analyte amperometric biosensors concentration by mixing precise mole fractions of KH2 PO4 and
relying on simple potentiostatic control. A new electronics sensing K2 HPO4 in order to achieve a pH value of 7.10. All reagents
methodology is required whereby the binary nature of the chemical employed in the abdominal trauma (ABT) gate were prepared in
output can be exploited. Moreover, the enzyme logic architecture this buffer solution. MG was employed as a mediator to enable the
eliminates the requirement for scaling the number of sensing ele- low-potential oxidation of NADH.
ments with the number of chemical analytes under investigation;
the fluctuations in multiple biochemical markers can be integrated 2.2. Electronic components
and processed to yield a single output, which can be monitored
by a single electronic sensing element. This property can, in turn, A linear voltage regulator (LP3990), switched capacitor volt-
alleviate the power consumption burden associated with multiple age converter (LM2664), quad micropower precision amplifier
sensing components. Thus far, however, microelectronic systems with CMOS input (LMP2234), and micropower comparator with
have not been adapted to meet the unique demands of digital sen- CMOS input (LPV7215) were procured from National Semiconduc-
sors that exploit biocomputing principles. tor (Santa Clara, CA). Single CMOS two-input AND (74LX1G08) and
In this study, we describe the design, development, and evalu- NAND (74LX1G00) gates were obtained from STMicroelectronics
ation of a new class of electronic systems specifically configured (Geneva, Switzerland). A CR1025 3 V manganese dioxide lithium-
to harness the bioprocessing capabilities of biomolecular logic ion coin cell battery was purchased from Panasonic Corp. (Osaka,
systems and to provide amperometric transduction of signals Japan). All other passives (resistors, potentiometers, capacitors,
generated by enzyme logic biosensors. The multivariate and ver- LEDs, switch, and battery holder) were acquired from Digikey Corp.
satile sensing capabilities of the concept are demonstrated, taking (Thief River Falls, MN). Block-level and circuit-level diagrams are
clinically relevant scenarios corresponding to combat injuries as illustrated in Fig. 1A and B, respectively.
a model. This biosensor is evaluated towards the amperomet- The linear voltage regulator was configured to generate a +1.8 V
ric determination of pathological levels of creatine kinase/lactate supply rail from the 3 V battery, which was fed into the switching
dehydrogenase and lactate/lactate dehydrogenase for the diagnosis voltage converter, thereby yielding a −1.8 V rail to implement fully
of soft tissue injury (STI) and abdominal trauma (ABT), respectively. differential voltage compliance at the potentiostatic unit. A NAND
The sensor employs enzyme cascades that imitate the operational gate was employed for the STI experiments in order to invert the
functionality of NAND (STI) and AND (ABT) logic gates in connection logic output generated by the comparator and consequently drive
with the detection of the biocatalytically processed chemical infor- the status indicator LED. For the ABT experiments, an AND gate
mation via disposable carbon screen printed electrodes (SPE). The was used in the place of the NAND gate to drive the status indicator
biosensor system enables a clinically relevant switching threshold LED. The selection of resistors employed in the potentiostat was as
to be pre-programmed into the device and configured as needed follows: R1 = R4 = R6 = R8 = 1 M, R2 = 1 k, R3 = 43 k, R5 = 100 k,
for the intended injury/application. The results presented clearly and R7 was adjusted in accordance with the switching threshold
indicate the potential of the new concept for the unequivocal iden- required by the application. The selection of capacitors employed
tification of pathophysiological conditions. It is anticipated that a in the potentiostat was as follows: C1 = C2 = 1 ␮F (used for regulator
user-friendly bioelectronic sensing system, such as the one dis- stability), C3 = C4 = 3.3 ␮F (switched converter charge storage), and
cussed here, would be well-suited to empower a non-technical C5 = 1 ␮F (low-pass filtering).
operator with the ability to identify a wide array of chemical agents A 19 × 19 mm 4-layer printed circuit board (PCB) was custom-
of importance in various clinical, security, and environmental sce- designed using an AutoCAD® electrical layout editor and
narios. outsourced for fabrication. The PCB consisted of separate power
and ground planes as well as a battery holder on the reverse side. A
2. Materials and methods digital logging multimeter was employed for the electrical mea-
surements. Photographs of the complete microelectronic device
2.1. Preparation of chemicals and reagents and its components are shown in Fig. 2A and B, respectively.

Potassium phosphate monobasic (KH2 PO4 ), potassium phos- 2.3. Electrode design and fabrication
phate dibasic (K2 HPO4 ), glycyl-glycine (Gly-Gly), magnesium
acetate tetrahydrate (MgAc), potassium hydroxide (KOH), bovine The fabrication of the ceramic-based screen-printed electrode-
serum albumin (BSA), creatine (CRTN), adenosine 5 -triphosphate sensor is detailed: A laser-scribed alumina substrate was obtained
disodium salt hydrate (ATP), phosphoenolpyruvic acid monopotas- from CoorsTek Inc. (Golden, CO). An Ag/AgCl-based ink from Ercon
sium salt (PEP), L(+)-lactic acid (LAC), ␤-nicotinamide adenine Inc. (E2414) was employed to define the conductive underlayer
dinucleotide reduced dipotassium salt (NADH), nicotinamide ade- as well as the reference electrode and printed directly onto the
nine dinucleotide (NAD+ ), methylene green (MG), pyruvate kinase substrate. A carbon-based ink (Ercon E3449) was then overlaid on
(PK) from rabbit muscle (E.C. 2.7.1.40), creatine kinase (CK) from the conductor to define the working and counter electrode geom-
rabbit muscle (E.C. 2.7.3.2), and lactate dehydrogenase (LDH) from etry. Finally, an insulator ink (Ercon E6165) was overlaid on the
porcine heart (E.C. 1.1.1.27) were purchased from Sigma-Aldrich Ag/AgCl and carbon layers to insulate all but the contact pads and
(St. Louis, MO) and were used as supplied without any pretreatment the upper segment of the electrodes. In each of the three afore-
or purification. Ultra pure deionized water (18.2 M-cm) supplied mentioned processing steps, a Speedline Technologies MPM-SPM
from a Barnstead Nanopure Diamond source (Waltham, MA) was screen printer was used to print the pattern onto the ceramic sub-
used in all experiments. strate using a custom-designed stainless steel stencil. Subsequent
208 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213

Fig. 1. (A) Process flow diagram outlining the equivalent functional behavior of the microelectronic sensing system and (B) the circuit-level schematic of the supporting
electronics designed for the analysis of abdominal trauma. In order to realize correct logic operation, the CMOS AND logic gate in the figure is replaced with a CMOS NAND
logic gate for the readout of the soft tissue injury system.

to the printing process, the patterned substrate was cured in a elevated under circumstances where muscular exertion, fatigue,
temperature-controlled convection oven (SalvisLab Thermocenter) injury, and trauma are sustained [12]. CK, a specific indicator of
at 120 ◦ C for 20 min and cleaved into test strips for single use. Each rhabdomyolysis, has been shown by Kaste et al. [13] to increase
screen printed three-electrode strip consisted of a circular carbon from an average serum level of 100 U/L under normal physiolog-
working electrode (geometrical area: 3 mm2 ) inscribed in a hemi- ical conditions to around 710 U/L when an STI event has been
spherical counter (area: 10 mm2 ) and reference electrode (area: incurred. Likewise, circulating levels of LDH, an enzyme frequently
2 mm2 ). employed for the determination of tissue breakdown and hemol-
ysis, can increase markedly from around 150 U/L under normal
2.4. Selection of the biomarkers and clinical relevance circumstances to over 1000 U/L under pathological states [14,15].
Abdominal trauma (ABT), whether of the penetrating or blunt
Among the plethora of relevant biomarkers implicated in soft variety, represents a common class of combat injury whereby one
tissue injury (STI), serum levels of CK and LDH become noticeably or multiple organs in the abdominal cavity are ruptured or oth-

Fig. 2. (A) Image of the microelectronic system (US 1¢ and screen printed three-electrode strip shown for size comparison). (B) Obverse and reverse detail of the microelectronic
system indicating the locations of the constituent components on the PCB.
 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213 209

erwise damaged [16]. In such scenarios, serum LAC and LDH are (with gain A) and supplies current through the counter electrode.
among the biomarkers of choice in the clinical setting when assess- Upon sensing a voltage generated at the reference electrode, OA2,
ing organ damage and malfunction [17,18]. Whereas LDH exhibits a voltage follower/buffer, syncs sufficient current through R2 in
a similar concentration profile as indicated above under this class order to maintain its output voltage at the input (VRE ) value. In
of injury [15], LAC has been shown to increase by Hara et al. from turn, VX is adjusted and the output potential/current of OA1 is
1.6 mM to 6.0 mM [14,15]. modified accordingly. The control amplifier thus functions as a
voltage-controlled current source that delivers sufficient current to
maintain the reference electrode at constant potential and arbitrate
2.5. Composition of the logic gates and protocol
the electrochemical reaction. In implementing negative feedback,
it is imperative that OA1 be able to swing to extreme potentials
In both the systems under investigation (STI and ABT), the nor-
to allow full voltage compliance required for chemical synthe-
mal physiological concentrations of the selected biomarkers were
sis. Furthermore, it is crucial that OA2 possesses very high input
employed as digital ‘0’ input signals, while the elevated patho-
impedance in order to draw negligible current; otherwise the refer-
logical concentrations were defined as ‘1’ input signals. Thus, the
ence electrode may deviate from its intended operating potential. In
systems were evaluated at four different combinations of the input
practice, the use of precision instrumentation amplifiers possessing
signals: (0,0), (0,1), (1,0), and (1,1), where only the last combina-
20 fA of input bias current enables unabated operation to the sub-
tion corresponded to pathological scenarios, while the three other
picoampere level, which is suitable for nearly all electrochemical
logic combinations reflected normal conditions or other irrelevant
studies.
physiological anomalies. In addition to the binary levels of the input
Employing the equivalent circuit model of the electrochemical
injury biomarkers, other reagents were experimentally optimized
cell, the current through the cell may be expressed in the frequency
and employed at constant concentrations. These supporting chem-
domain (ω) as
icals served as the system “machinery” and therefore performed
the biochemical analysis of the logic input signals. AVX − VWE
iCELL (ω) = , (1)
The STI experiments were conducted by employing 0.3 mM RC + (RW /(1 + jωRW CW ))
NADH, 0.5 mM PEP, 2 mM ATP, 15 mM CRTN, 0.3 mM MG, 2000 U/L
and the voltage established at the reference electrode is given by
PK, 100 U/L (‘0’)/710 U/L (‘1’) CK, and 150 U/L (‘0’)/1000 U/L (‘1’) LDH
the relation
in a 50 ␮L sample volume. All reagents were mixed in a tube and  RW

subjected to a 180-s incubation at 37 ◦ C in a heatblock. Following VRE (ω) = VWE + iCELL , (2)
this incubation period, the solution was dispensed on the elec- 1 + jωRW CW
trode surface and a chronoamperogram was subsequently initiated where icell represents the current flowing from the counter elec-
whereby a working electrode potential of 0.0 V (vs. Ag/AgCl) was trode to the working electrode. The voltage at the counter electrode
maintained for 60 s. will follow the potential seen at node VX ,
The ABT experiments were conducted by employing 10 mM
( − 1)VWE − (R2 /R1 )V +
NAD+ , 1 mM MG, 1.6 mM (‘0’)/6.0 mM (‘1’) LAC, and 150 U/L VCE (ω) = AVX = A , (3)
(‘0’)/1000 U/L (‘1’) LDH in a 50 ␮L sample volume. All reagents were (A − 1 − (R2 /R1 ))
mixed in a tube and subjected to a 180-s incubation at 37 ◦ C in a and
heatblock. Following this incubation period, the solution was dis- 1
pensed on the electrode surface and a chronoamperogram was = . (4)
1 + (RC /RW )(1 + jωRW CW )
subsequently initiated whereby a working electrode potential of
0.0 V (vs. Ag/AgCl) was maintained for 60 s. The potential at the working electrode (with respect to the
It should be noted that references to digital logic gates in bold reference) must be specified as it is a crucial parameter in electro-
typeface (i.e. NAND) represent enzyme-based manifestations of chemistry that dictates the activation of the electroactive species.
logic gates. On the other hand, references without a bold typeface More specifically, the application of a suitable potential at the work-
(i.e. NAND) represent their CMOS counterparts. ing electrode will ensure that the electroactive substance within
the medium is oxidized or reduced. Consequently, this will yield a
Faradaic current proportional to the concentration of the analyte by
3. Results and discussion the Cottrell equation [22]. Synthesizing the above expressions, net-
work analysis may be performed, yielding the frequency-domain
3.1. Design of the electronic backbone voltage at the working electrode,
−R2 R3
The new electronic architecture has been designed to control VWE (ω) = V+ (5)
biocomputing systems applied to diagnostic merits. To simplify R1 (R3 + (RW /(1 + jωRW CW )))
analysis, a Randles–Ershler equivalent R-C circuit model [19–21] and the DC response can be evaluated
is employed to emulate the electrical behavior of the electro-
−R2 R3
chemical system, as displayed in Fig. 1B. This model consists of a VWE (DC) = V +. (6)
R1 (R3 + RW )
parallel capacitor (CW , corresponding to the double layer capaci-
tance arising from the accumulation of a net surface charge at the The above relations indicate that, for a system with RW  R3 , the
working electrode) and resistor (Rw , corresponding to the charge potential at the working electrode can be adjusted by modifying
transfer/Faradaic resistance arbitrated by the electroactive species) the ratio between R2 and R1 . More crucially, Eqs. (5) and (6) eluci-
in series with another resistor (RC , the total solution resistance) date that the working electrode voltage is inversely proportional to
[19,22]. The potentials at the working, counter, and reference elec- the Faradaic resistance and therefore directly proportional to the
trodes are denoted as VWE , VCE , and VRE , respectively. The positive Faradaic current arising from the electrochemical reaction. Accord-
supply voltage is denoted as V+ . ingly, by the Cottrell equation, the concentration of the analyte can
As illustrated in Fig. 1B, the potentiostatic unit consists of two be extrapolated and should be linearly related to the signal arising
LMP2234 precision instrumentation operational amplifiers (OA) at the working electrode. It is important to note that R3 is selected
configured in the following arrangement: control amplifier OA1 to enable best noise performance at the expense of response time.
amplifies the differential voltage seen between node VX and ground Increasing this value will enable lower noise readings, but longer
210 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213

Fig. 3. (A) Biocatalytic cascade instigated by creatine kinase (CK) and lactate dehydrogenase (LDH) emulating NAND operation, (B) the equivalent logic system, and (C) the
corresponding truth table with biomedical conclusions drawn from the combinations of the input signals.

response times. For quasi-real-time measurements where a read- an LED. An AND logic gate is employed when the enzyme logic
ing is recorded on a non-continuous basis at some fixed interval, gate implements the AND operation. Accordingly, when the output
it is appropriate to employ a moderate R3 resistance in order to of the potentiostat and supporting analog subsystem exceeds the
enable the highly sensitive detection of the analyte. pre-programmed threshold level, the comparator outputs a ‘high’
OA3, an integrator (another LMP2234 precision instrumentation (logical ‘1’) voltage, hence driving the output AND gate high and
operational amplifier), implements a low-pass filtering operation thereby illuminating the LED. Likewise, a NAND logic gate is utilized
and provides low-noise gain to the signal arising at VWE . R4 provides when the enzyme logic gate implements the NAND operation. In
the necessary feedback at DC/low frequencies (where C5 has large this case, the presence of a sufficient level of analyte would cause
reactance) to maintain a stable output at the correct value. With the output of the potentiostat and supporting analog subsystem to
suitable choice of R4 and C5 , the integrator can mitigate the high- fall below the pre-programmed threshold level. As a consequence,
frequency oscillation/instability induced by the capacitive loading the output of the comparator would fall to the ground potential
of the potentiostat. The output of the integrator is subsequently (logical ‘0’), hence driving the output of the NAND gate high and
amplified by OA4 (LMP2234), a non-inverting voltage amplifier, in resulting in the illumination of the LED.
order to provide additional gain to bring the signal to rail levels. With the above implementation of the electronic hardware,
The output voltage of the final amplifier stage is given by the complete sensor system consumed 218 ␮A of current at 3.0 V,

R2 (R4 /(1 + jωR4 C5 ))

 R6
 1
and thus the total power dissipation was 654 ␮W. Given a typi-
V0 (ω) = 1+ V + , ωc = cal 30 mAh 3 V CR1025 coin cell battery, such a system could be
R1 (R3 + (RW /(1 + jωRW CW ))) R5 R4 C5 sustained for over 45 h under continuous use.
(7)

and 3.2. High-fidelity readout of soft tissue injury

R2 R4
 R6

V0 (DC) = 1+ V +. (8) With a robust electronic backbone in place, the micro-
R1 (R3 + RW ) R5
/bioelectronic sensor system was applied towards the detection
As can be deduced from Eqs. (7) and (8), the output voltage of of STI with an enzyme-based NAND gate. Fig. 3A illustrates the
OA4 is inversely proportional to the Faradaic resistance and there- biocatalytic cascade whereby the enzyme inputs CK and LDH are
fore directly proportional to the Faradaic current arising from the processed to yield NADH as an output. The equivalent logic gate is
electrochemical reaction. The output voltage V0 thus serves as an shown in Fig. 3B. Upon the detection of abnormally high levels of
indicator of the amount of electroactive analyte present in the sys- both CK and LDH, the quantity of NADH present in the chemical
tem. system would decrease, as is evident from the truth table shown in
Following the analog signal processing, V0 is incident on a com- Fig. 3C, thereby triggering the illumination of the LED.
parator, which compares this value with a pre-established voltage In order to resolve the proper switching threshold that would
VT that is implemented by adjusting the potentiometer R7 in rela- indicate the occurrence of an STI event, the sensor was evaluated
tion to a fixed resistor R8 . In the event that V0 exceeds VT , the towards the operation of the NAND gate under four input logic
comparator will output the full rail voltage (logical ‘1’); otherwise, combinations. Fig. 4A displays a bar chart obtained at the SPE by
the output of the comparator will be at ground potential (logical the NAND gate upon the application of all four of the input logic
‘0’). In this manner, the device operates as a 1-bit analog-to-digital combinations for three independent trials. At 60 s sampling time,
converter with an adjustable switching threshold. the difference in mean voltage between the pathological logic level
The output of the comparator is channeled to one of the inputs of (1,1) and the physiological logic level in closest proximity (1,0) was
a CMOS logic gate and the other input is tied to the supply voltage. 0.898 V. An exceptionally low standard deviation of less than 90 mV
The CMOS logic gate serves to source sufficient current to drive was maintained at every logic level.
 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213 211

Fig. 4. (A) Bar chart featuring the NAND logic operation for the corresponding combinations of input signals. Electrochemical measurements were performed at E = 0.0 V vs.
Ag/AgCl. Dashed lines indicate the decision threshold for the realization of NAND gate operation. (B) Images of the microelectronic system under the application of various
combinations of the input biomarkers CK and LDH. Only the pathological scenario involving high levels of both CK and LDH corresponding to the (1,1) logic level rendered
an output logic 0, resulting in the illumination of an LED.

Given the need to institute a threshold for the presentation of dard deviation of less than 60 mV was maintained at every logic
an affirmative diagnosis, the decision threshold was established as level.
the midway point between the (1,1) and (1,0) logic levels, 0.535 V. In order to achieve the highest-fidelity diagnosis possible, the
As such, potentiometer R7 was adjusted to 297 k and accordingly decision threshold was established at the halfway point between
a voltage divider (with respect to R8 ) was implemented to realize a the (1,1) and (0,1) logic levels, 1.254 V. As such, potentiometer R7
reference voltage (0.535 V) for the comparator. In pathophysiolog- was adjusted to 697 k and accordingly a voltage divider (with
ical circumstances that resulted in an output voltage V0 below this respect to R8 ) was implemented to realize a reference voltage
threshold voltage VT , light emission from the LED ensued. Fig. 4B (1.254 V) for the comparator. In pathophysiological scenarios that
displays photographs of the sensor under the application of the resulted in an output voltage V0 exceeding this threshold voltage VT ,
(0,0), (0,1), (1,0), and (1,1) logic levels once the programmable light emission from the LED ensued. Fig. 6B displays photographs
threshold was established. Clearly, only the pathological case (1,1)
resulted in the illumination of the LED, thereby alerting the opera-
tor that an STI event has occurred and demonstrating the system’s
unambiguous assessment of the pathophysiological state.

3.3. High-fidelity readout of abdominal trauma

Following the system-level validation of the micro-

/bioelectronic sensor towards the evaluation of STI, the sensor
was subsequently applied towards the detection of ABT with an
enzyme-based AND gate. The biocatalytic cascade is displayed in
Fig. 5A whereby the input biomarkers LAC and LDH are processed
to yield NADH as an output. The equivalent logic gate is shown
in Fig. 5B. In contrast to the STI system, upon the detection of
abnormally high levels of both LAC and LDH, the quantity of NADH
present in the chemical system would increase. This trend can be
inferred from the truth table shown in Fig. 5C, and this process can
be monitored by the operator via an LED display.
As in the STI system, in order to resolve the proper switch-
ing threshold that would indicate the occurrence of an ABT event,
the sensor was evaluated towards the operation of the AND gate
under four input logic combinations. Fig. 6A displays a bar chart
obtained at the SPE by the AND gate upon the application of all
four of the input logic combinations for three independent trials.
Fig. 5. (A) Biocatalytic cascade instigated by lactate (LAC) and lactate dehydrogenase
At 60 s sampling time, the difference in mean voltage between the
(LDH) emulating AND operation, (B) the equivalent logic system, and (C) the corre-
pathological logic level (1,1) and the physiological logic level in sponding truth table with biomedical conclusions drawn from the combinations of
closest proximity (0,1) was 0.267 V. An exceptionally low stan- the input signals.
212 J.R. Windmiller et al. / Sensors and Actuators B 155 (2011) 206–213

Fig. 6. (A) Bar chart featuring the AND logic operation for the corresponding combinations of input signals. Electrochemical measurements were performed at E = 0.0 V vs.
Ag/AgCl. Dashed lines indicate the decision threshold for the realization of AND gate operation. (B) Images of the microelectronic system under the application of various
combinations of the input biomarkers LAC and LDH. Only the pathological scenario involving high levels of both LAC and LDH corresponding to the (1,1) logic level rendered
an output logic 1, resulting in the illumination of an LED.

of the sensor under the application of the (0,0), (0,1), (1,0), and /bioelectronic sensing concept is the first example, to the best of
(1,1) logic levels once the programmable threshold was established. our knowledge, of the development of an electronic system specif-
Clearly, only the pathological case (1,1) resulted in the illumina- ically tailored for the evaluation of biocomputing systems applied
tion of the LED, thereby alerting the operator that an ABT event has to diagnostic merits. The low-power, low-cost, and miniaturized
occurred and again demonstrating the system’s diagnostic integrity embodiments of the sensor system make the design particularly
and utility as a versatile backbone for the readout of enzyme logic attractive for diverse field operations. With further development
gates. of the supporting microelectronic systems, the sensor system
It is anticipated that the two systems presented here will would empower the non-technical end-user with the ability to
function as intended for a majority of the population in circum- assess the presence of chemical species in various clinical, security,
stances where the biomarker levels fall within clinically established and environmental scenarios in a straightforward and convenient
ranges. However, in both scenarios, owing to the variable extent manner.
of afflictions and the presence of a myriad of sources of potential
interference, the execution of a large-scale clinical investigation Acknowledgement
that integrates various degrees of injury is imperative in order to
select the most optimal decision threshold level for the population This work was supported by the Office of Naval Research (Award
at large. #N00014-08-1-1202).

4. Conclusions References

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biomarkers for the diagnosis of soft tissue injury, Sens. Actuators B 150 (2010) Joshua Ray Windmiller received the B.Sc. and M.Sc. in Electrical Engineering in
285–290. 2007 and 2009, respectively, from the Jacobs School of Engineering at the Univer-
[10] D. Melnikov, G. Strack, J. Zhou, J.R. Windmiller, J. Halámek, V. Bocharova, M.C. sity of California, San Diego. He is currently pursuing the Ph.D. degree in Electrical
Chuang, P. Santhosh, V. Privman, J. Wang, E. Katz, Enzymatic AND logic gates Engineering at the UCSD Jacobs School where his research focuses on the develop-
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Padmanabhan Santhosh received the Ph.D. degree in Industrial Chemistry from
Chuang, J. Zhou, P. Santhosh, G.V. Ramirez, M.A. Arugula, J. Wang, E. Katz, Multi-
Alagappa University, India. He did postdoctoral work at Kyungpook National Uni-
enzyme logic network architectures for assessing injuries: Digital processing
versity, Korea, and was scientist at Max-Planck-Institute for Solid State Research,
of biomarkers, Mol. BioSyst. 6 (2010) 2554–2560.
Stuttgart, Germany. He has published more than 55 research articles in peer-
[12] J.E. Olerud, L.D. Homer, H.W. Carroll, Incidence of acute exertional rhabdomyol-
reviewed journals and has 3 patents. His research interests include biosensors and
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enzyme based logic systems.
Int. Med. 136 (1976) 692–697.
[13] M. Kaste, J. Hernesniemi, H. Somer, M. Hillbom, A. Konttinen, Crea- Evgeny Katz received his Ph.D. in Chemistry from Frumkin Institute of Electro-
tine kinase isoenzymes in acute brain injury, J. Neurosurg. 55 (1981) chemistry (Moscow), Russian Academy of Sciences, in 1983. He was a senior
511–515. researcher in the Institute of Photosynthesis (Pushchino), Russian Academy of Sci-
[14] A. Kratz, M. Ferraro, P.M. Sluss, K.B. Lewandrowski, Laboratory reference values, ences, in 1983–1991. In 1992–1993 he performed research at Technische Universität
N. Engl. J. Med. 351 (2004) 1548–1563. München (Germany) as a Humboldt fellow. Later, in 1993–2006, Dr. Katz was a
[15] I. Hara, Y. Nakano, H. Okada, S. Arakawa, S. Kamidono, Treatment of crush syn- research associate professor at the Hebrew University of Jerusalem. From 2006 he
drome patients following the great Hanshin earthquake, Int. J. Urol. 4 (1997) is Milton Kerker Chaired Professor at the Department of Chemistry and Biomolec-
202–205. ular Science, Clarkson University, NY (USA). He has (co)authored over 300 papers
[16] J.J. Davis, I. Cohn, F.C. Nance, Diagnosis and management of blunt abdominal and holds 20 international patents. He serves as an Editor-in-Chief for IEEE Sen-
trauma, Ann. Surg. 183 (1976) 672–678. sors Journal. His scientific interests are in the areas of bioelectronics, biosensors
[17] A.K. Malhotra, R. Latifi, T.C. Fabian, R.R. Ivatury, S. Dhage, T.K. Bee, P.R. Miller, and biofuel cells. Currently he is actively involved in the research in biocomputing,
M.A. Croce, J.A. Yelon, Multiplicity of solid organ injury: influence on man- signal-responsive materials and their applications in logically operating biosensors.
agement and outcomes after blunt abdominal trauma, J. Trauma 54 (2003)
925–929. Joseph Wang received Ph.D. from Israel Institute of Technology in 1978. From 1978
[18] K.K. Tan, S.L. Bang, A. Vijayan, M.T. Chiu, Hepatic enzymes have a role in the to 1980 he served as a research associate at the University of Wisconsin (Madi-
diagnosis of hepatic injury after blunt abdominal trauma, Injury 40 (2009) son) and joined New Mexico State University (NMSU) at 1980. In 2001–2004, he
978–983. held a Regents Professorship and a Manasse Chair positions at NMSU, and served as
[19] E. Barsoukov, J.R. Macdonald, Impedance Spectroscopy: Theory, Experiment, the director of Center for Bioelectronics and Biosensors of Arizona State University
and Applications, Wiley, 2005. (ASU). Currently, he is Professor in Department of Nanoengineering at University of
[20] J.E.B. Randles, Kinetics of rapid electrode reactions, Discuss. Faraday Soc. 1 California, San Diego (UCSD). Dr. Wang has published more than 800 papers and he
(1947) 11–19. holds 12 patents. He became the most cited electrochemist in the world and received
[21] B.V. Ershler, Investigation of electrode reactions by the method of charging- the 4th place in the ISI’s list of ‘Most Cited Researchers in Chemistry’ in 1996–2006.
curves and with the aid of alternating currents, Discuss. Faraday Soc. 1 (1947) Since 1980, 20 Ph.D. candidates and 75 research associates have studied with him.
269–277. He is the Editor-in-Chief of Electroanalysis. His scientific interests are concentrated
[22] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applica- in the areas of biosensors, bioelectronics, bionanotechnology and electroanalytical
tions, 2nd ed., Wiley, 2000. chemistry.