IEEE ELECTRON DEVICE LETTERS, VOL. 27, NO. 7, JULY 2006
595
A CMOS, Fully Integrated Sensor for Electronic
Detection of DNA Hybridization
Massimo Barbaro, Annalisa Bonfiglio, Luigi Raffo, Member, IEEE,
Andrea Alessandrini, Paolo Facci, and Imrich Barák
Abstract—An integrated field-effect device for fully electronic
deoxyribonucleic acid (DNA) detection was realized in a standard
CMOS process. The device is composed of a floating-gate MOS
transistor, a control-capacitor acting as integrated counterelectrode, and an exposed active area for DNA immobilization. The
drain–current of the transistor is modulated by the electric charge
carried by the DNA molecules. After DNA hybridization, this
charge increases and a change in the output current is measured.
Experimental results are provided. Full compatibility with a standard CMOS process opens the way to the realization of low-cost
large-scale integration of fast electronic DNA detectors.
Index Terms—CMOS biosensor, deoxyribonucleic acid (DNA)
chip, fully electronic DNA detection.
Fig. 1. Structure of the device.
I. I NTRODUCTION
T
HE STANDARD commercial approach to deoxyribonucleic acid (DNA) hybridization detection is based on the
use of fluorescent, radioscope, and other labels and can be
schematically summarized in the following procedure: 1) A
probe of single-stranded known sequence of DNA is immobilized on a substrate; 2) the unknown sequence (target) is labeled
with a specific tag; 3) when hybridization occurs, the target
sequence binds to its complementary strand immobilized on
the surface; and 4) its presence can be optically detected [1].
The required instrumentation is bulky, costly, and not portable
[2]. For this reason, a number of new approaches for direct
label-free detection of DNA hybridization have been proposed
in the last decade, among them are detection based on quartz
crystal microbalance (QCM) [3], the cantilever-based techniques [4], and several examples of electronic detection method
[5]–[8]. Direct electronic detection has several advantages with
respect to other approaches: The detector is incorporated in
the substrate, the output signal can be directly acquired and
processed on a chip, and automatic recognition is achievable
in real time and at low cost. Moreover, electronic detection by
means of standard CMOS devices would pave the way to the
realization of simple, portable, inexpensive detection platforms,
Manuscript received January 25, 2006; revised April 18, 2006. The review
of this letter was arranged by Editor K. Kornegay.
M. Barbaro and L. Raffo are with the Department of Electrical and Electronic
Engineering, Istituto Nazionale per la Fisica della Materia (INFM)-University
of Cagliari, 09123 Cagliari, Italy.
A. Bonfiglio is with the Department of Electrical and Electronic Engineering,
INFM-University of Cogliari, 09123 Cogliari, Italy and also with nanoStructures and bioSystems at Surfaces-Istituto Nazionale per la Fisica della MateriaConsiglio Nazionale delle Ricerche (S3-INFM-CNR), 41100 Modena, Italy.
A. Alessandrini and P. Facci are with S3-INFM-CNR, 41100 Modena, Italy.
I. Barák is with the Institute of Molecular Biology, Slovak Academy of
Science, 81434 Bratislava, Slovak Republic.
Digital Object Identifier 10.1109/LED.2006.876303
exploiting the advanced technology of consumer electronics.
In this letter, we present a novel field-effect device for direct
electronic detection of DNA hybridization based on a standard
commercial CMOS process.
II. M ATERIALS AND M ETHODS
The proposed device, whose basic structure is shown in
Fig. 1, incorporates the characteristics of floating-gate transistors used in Flash memories and those of gate-exposed transistors such as the ion-sensitive field-effect transistor (ISFET)
[9] or the chemically modified FET (CHEMFET). In fact, it is
composed of a transistor M0 , a poly1–poly2 control capacitor
CC , an active area AS , and a parasitic capacitor CF between the
floating gate and silicon body. The two layers of polysilicon are
commercially available in CMOS processes with analog option,
whereas direct access to the surface of the gate is obtained
opening a test pad in the middle of the die and connecting the
topmost exposed aluminum layer with the gate by means of
available routing layers. The active area is the site for DNA
immobilization and detection after proper chemical activation
of the surface by means of deposition of a spacer layer capable
of anchoring DNA molecules. The working principle has been
described, modeled, and simulated in [10] and can be summarized with the following output equation:
VTHF = VTH0 −
QDNA + Q0
CC + CF
(1)
where VTHF is the effective threshold voltage of the transistor,
VTH0 is the native threshold, Q0 is the electric charge initially
trapped in the floating gate, and QDNA is the total charge of
DNA molecules. Equation (1) states that effective threshold
voltage of the transistor is modulated by a term that is proportional to the amount of net electric charge immobilized on the
0741-3106/$20.00 © 2006 IEEE
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IEEE ELECTRON DEVICE LETTERS, VOL. 27, NO. 7, JULY 2006
Fig. 2. Microphotograph of the chip packaged with the PDMS microchannel
structure.
active area, thus an hybridization event can be detected by a
shift in such voltage.
A test chip was realized in a standard 0.8 µm CMOS
process by AustriaMicroSystems (AMS). The chip, shown
in Fig. 2, hosts 16 sensors with four different sizes for the
active area (ranging from 40 × 40 to 100 × 100 µm2 ). Each
chip was directly bonded to an electronic board, obtaining
a very compact test device whose dimension is the same
of a microscope coverslip. The 16 sensors were subdivided
in two clusters to test the device detection capability. The
sensing surfaces of a reference cluster (T0, sensors 8–15)
were activated with a reference probe oligonucleotide designed
with 13-mer spacer, followed by a specific sequence from
variable region of 16S recombinant DNA (rDNA) of bacterium Bacillus subtilis. The oligonucleotide sequence was
5 -(T)13 GGTTTCCGCCCCTTAGTG-3 . The second cluster
(T1, sensors 0–7) was activated with a different oligonucleotide
with sequence 5 -(A)13 TCGGTGTAAAGGCTCT-3 . The hybridization test was performed by exposing the surface of
the devices to two different solutions containing the complementary sequences of each oligonucleotide (targets) dissolved in a low ionic strength buffer (to minimize electrical
noise). Surface activation procedure has been reported elsewhere [11] and required two steps: 1) deposition of a layer
of properly functionalized spacer molecules on aluminum pad
and 2) anchoring of the DNA single strands on the spacer.
To this aim, the native aluminum oxide was silylated with
(3-mercaptopropyl)trimethoxysilane (3-MPTS), which exposes
a thiol group. The oligonucleotides were modified by inserting another thiol group at 5 position. In this way, covalent
immobilization takes place via the formation of intermolecular
S–S bonds between the thiolate single-stranded DNA (SSDNA)
and the surface SH moieties, obtaining a layer of SSDNA
molecules. During DNA hybridization test, the two clusters of
devices have been inserted in two reaction chambers that have
been built on the silicon chip by means of polydimethylsiloxane
(PDMS), an elastomer that is normally employed for building
microfluidic systems [12]. DNA solutions entered the two small
chambers flowing from inlet to outlet channels, filled by two
syringes. During hybridization steps, the chip was inserted
in a temperature-controlled chamber to keep its temperature
at the value requested for performing the reaction (50 ◦ C).
Fig. 3. Differential threshold voltages measured for each pair of active reference transistors after each of the following events: START (functionalization
with 3-MPTS), STEP_T1 (immobilization of T1), STEP_T0 (immobilization
of T0), STEP_P1 (injection of P1), and STEP_P0 (injection of P0). Differential
threshold voltage is given by OUT[i] = VTHF[i] − VTHF[i + 8].
The electrical characterization of the device was performed by
means of a custom board hosting circuitry for biases, analog-todigital conversion, and data acquisition on a personal computer.
To test the capability of detecting DNA hybridization
processes, the following sequence of steps was performed:
“START” (functionalization of the chip surface with 3-MPTS),
“STEP_T1” (immobilization of T1 oligonucleotide on the active areas of a cluster of sensors), “STEP_T0” (immobilization of T0 oligonucleotide on a second cluster), “STEP_P1”
(injection on the entire chip surface of the P1 oligonucleotide,
complementary to T1), and “STEP_P0” (injection on the entire
chip surface of the P0). After each step, the electrical characteristics of each MOS transistor were measured applying a
voltage to the control gate, and the effective threshold voltage
was extrapolated.
III. R ESULTS AND D ISCUSSION
Our first measurements have concerned the testing of a single
device. As foreseen, the threshold voltage of each device is
extremely sensitive to any charge variation in the environment
surrounding the device. To tradeoff such high sensitivity with
selectivity, nonspecific charge contributions were eliminated
by adopting a differential approach. A differential threshold
voltage was calculated subtracting the computed threshold of
each sensor in the reference cluster (T0, sensors 8–15) from
the threshold of the corresponding sensor in the active cluster (T1, sensors 0–7). The differential threshold is given by
OUT[i] = VTHF[i] − VTHF[i + 8], where 0 ≤ i ≤ 7. In this
way, changes in the threshold due to a global cause (such as the
rinsing procedure) affect all the sensors at the same time and are
cancelled by differentiation. On the contrary, a change specific
to one sensor (such as successful hybridization) can be detected
as a differential signal. Initial offset due to large mismatch
of floating-gate devices was cancelled off-line by subtracting
from the outputs at each step the outputs from the previous
steps. The experimental results are shown in Fig. 3. The results
confirm what is predicted by (1): When the total amount of
BARBARO et al.: A CMOS, FULLY INTEGRATED SENSOR FOR ELECTRONIC DETECTION OF DNA HYBRIDIZATION
DNA molecules increases on a specific sensor, its threshold
voltage increases with respect to the corresponding sensor of
the reference cluster. In this way, hybridization can be easily
detected by the sign of the plotted differential threshold, in
which a positive sign represents recognition of P1 probe, which
hybridizes with its complementary DNA and thus increases the
threshold voltages of transistors of T1 cluster with respect to
those of T0 cluster. Following this procedure, we were able to
detect completion of hybridization reaction with all eight pairs
of sensors for target P1 and with five out of eight pairs for
target P0 (OUT7 is undecided). The nonuniform response of
the last step is probably due to the statistical fluctuations of the
biological reaction that is predictable only in a statistic sense.
It is in principle possible to minimize this effect by optimizing
the stringency of the coupling reaction, but this activity requires
a large number of refinement in the experimental. It may be
noted that the outputs seem not to be correlated to the size of the
different sensors: This is predicted by (1) because an increase of
the area increases CF at the denominator as well as QDNA at the
numerator, when the silane layer is dense and the concentration
of oligonucleotides is high.
In conclusion, we have designed, realized, and successfully
tested a standard CMOS device for detection of DNA hybridization reaction. This device is based on a simple detection
method that exploits the charge sensing capabilities of a floating
metal gate where a layer of SSDNA has been immobilized.
In this way, it was possible to obtain a very high sensitivity
to any charge variation occurring in the vicinity of the DNA
layer, including a hybridization event. With respect to other
devices based on an exposed gate [6]–[8], the use of the control
gate as an integrated counterelectrode makes the device fully
compatible with a standard CMOS process. Moreover, because
597
the operating point of the transistor is set by the control voltage,
the minimum amount of detectable DNA charge can be sensibly
lower than the minimum charge required to turn on the device.
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