Elsevier

Microelectronics Journal

Volume 37, Issue 12, December 2006, Pages 1610-1619
Microelectronics Journal

Design and characterization of novel read-out systems for a capacitive DNA sensor

https://doi.org/10.1016/j.mejo.2006.04.020Get rights and content

Abstract

This paper presents novel read-out electronic systems for a fast DNA label-less detection. The capacitive shift due to the hybridization effect is monitored by means of a charge sensitive amplifier and a differential stage. The systems provide an A/D conversion and an evaluation of the capacitive shift amount with a resolution of 11 bit. The read-out solutions demonstrate the ability to identify a 0.01% variation on the capacitive value of the sensor. The investigated techniques are suitable for monolithic systems or for a micro-fabricated array of sensors.

Introduction

Surface-based methods for the detection of biological molecules such as DNAs and proteins have revolutionized the biological detection. However, only limited use has been made of the electrical properties of biologically modified interfaces as a basis for biological sensing.

The wealth of available DNA sequence data makes it possible to identify many diseases as well as biological threats such as the presence of an infectious agent in the environment. Improving the sensitivity, selectivity, speed, simplicity and reducing the cost of such assays are important goals that will significantly affect the administration of health care at locations ranging from the patient's bedside to the battlefield.

Alterations in gene expression have profound effects on biological functions. These variations in gene expression are at the core of altered physiologic and pathologic processes. DNA array technologies provide the most effective means of identifying gene expression and genetic variations.

DNA is prepared from a wide variety of samples such as tissue, bacteria, saliva, etc. For genotyping analysis, the sample is genomic DNA. For expression analysis, the sample is cDNA, DNA copies of RNA. The DNA samples are tagged with a radioactive or fluorescent label and applied to the array. Single-stranded DNA will bind to a complementary strand of DNA. At positions on the array where the immobilized DNA recognizes a complementary DNA in the sample, binding or hybridization occurs. The labeled sample DNA marks the exact positions on the array where binding occurs, allowing automatic detection. The output consists of series of hybridization events, indicating the presence or the relative abundance of specific DNA sequences that are present in the sample.

Conventional arrays often rely on the detection of fluorescence from a molecular fluorophore. Electronic detection of hybridization is expected to require less complicated instrumentation and feature similar detection limits compared to the traditional optical methods.

When a conductor is placed in an electrolytic solution, a potential is generated due to an unequal distribution of charges across the interface. Two oppositely charged layers, one on the electrode surface and one inside the electrolyte form a “double layer,” which behaves as a parallel plate capacitor. When an additional layer is present at the electrode, such as an oxide or a self-assembled monolayer (SAM), an additional capacitance and/or resistance associated with that layer is added to the circuit. In principle, the introduction of molecules, such as DNA, to that interfacial region, will affect the measured complex impedance through changes of the local geometry, the dielectric constant, and the amount of charges at the electrode/electrolyte interface. A robust sensor must be able to measure the impedance change with a signal-to-noise ratio (S/N) that yields the desired sensitivity [1], [2], [3], [4].

A perfect bio-molecule recognition layer, for example a SAM of a single-stranded DNA (a SAM of ssDNA), would cover the electrode completely and the selective binding of complementary bio-molecules to that layer (i.e., hybridization in the given example) would be the only contributing impedance element.

Impedance-based sensors can give also a confuse detection in presence of non-selective binding. In order to remove the non-specific signal an experimental set-up including a reference sensor may be used. This sensor, functionalized, implemented in exactly the same way as the working sensor (except with non complementary DNA or a non-binding protein), is expected to have the same behavior as the working sensor towards non-specific interactions, and therefore to allow a specific differential measurement.

For label-less impedance sensing of bio-molecule binding both functionalized insulators and metals have been attempted.

The feasibility of using electrochemical impedance measurements on functionalized hetero-structures (semiconductor/dielectric/electrolyte) to directly detect hybridization between complementary homo-oligomer DNA strands without labels was demonstrated. When metal electrodes such as Pt and Au are used as the substrate for bio-layers they showed stronger signals. A typical example is the work by Bergren [4] who demonstrated the feasibility of a biosensor for direct detection of DNA hybridization on gold, but even here the detection capability was not very high and the reproducibility of the sensors was poor. Janata et al. [5], [6] show how small variations in the electrolyte could affect the impedance signal not because of a double-layer capacitance variation but because of electron transport resistance instability. The latter gives an indication of the typical signal level one can expect when detecting bio-molecules using a differential impedance method without the use of any label. With suitable signal amplification methods much higher impedance differences (orders of magnitude) can be obtained. Charge transfer effects always confound label-less impedance results and often dominate the total system impedance. To reduce this effect one must attempt to use a well-polarized electrode.

The ability to fabricate a perfectly polarized membrane [6] is still in an explorative phase. With the introduction of different grafting techniques such as SAMs or polypyrrole functionalization, it has been demonstrated that it is possible to reach a higher level of electrode polarization, avoiding the Faradic or diffusive current variations to a significant degree. Commercial products remain elusive though as reproducibility and selectivity remain major obstacles.

Recently a sequence specific DNA biosensor based on capacitance monitoring of the hybridization event has been developed.

The approach is based on the detection of capacitance changes produced by the DNA hybridization events onto the sensing interface. This approach is particularly suitable for the realization of a complete, single-chip solution through the integration of the sensing element with the micro-fluidic and electronic parts that complete the system.

The hybridization of targets increases the quantity of biological material that insulate the gold electrode from the electrolyte solution, hence the thickness of the capacitance dielectric. Furthermore, it changes also the relative dielectric constant of the insulating layer.

Variation of the sensor capacitance in the order of 30–50% are expected as result of DNA hybridization. However the sensing system capability depends on the appropriate choice of electronics readout. For the sensors geometry considered in our experiments, the sensor capacitance values can range between 20 nF down to 100 pF, depending on the electrodes area, solution concentration and biological material quantity.

Since it was realized a single board to measure different kinds of sensors, the electronics has been implemented such to follow the capacitive swing saving the needed linearity and reaching the desired resolution (>10 bit).

Moreover the readout system has been design to offer the possibility to perform an absolute capacitive measurement and also to compare the value of the changed capacitance of the sensor with the starting one to have a fast detection of the amount of the capacitive shift and then of hybridization occurrence.

In this paper, the focus is on the readout system for the novel sensor. The designed electronic system is able to detect the absolute value of the capacitance of the sensor with a resolution better than 1% and also the capacitance shift with a resolution better then 0.01%. The electronic systems described here is suitable for integration allowing a complete single-chip solution together with the micro-fabricated sensor.

Section snippets

Capacitive biosensor

Fig. 1 shows the principle scheme of the biosensor under study. In this case of interest [6], one of the gold electrodes of the sensing capacitors (A in Fig. 1) is covered with a oligonucleotides layer, realized by means of alkanethiols, and the interface exhibited ideal capacitive behavior in the 10–100 Hz frequency range (CPROBES in Fig. 1). Fig. 1 shows also in the bottom part the electric equivalent model as proposed by Randles [7], Cdl is the double layer capacitance shown at the

Read-out systems for the capacitive biosensor

Due to the peculiarity of the biosensor above described, read-out systems able to detect the occurrence of DNA hybridization and to quantify the amount of such event, recognizing in this way the target concentration, are required.

The hybridization determines the biosensor capacitance value reduction. The read-out system has to be able to detect capacitance shifts going from 30% to 50%.

To this end, three configurations for the read-out electronics are proposed in this paper. Each of them is able

Single-channel detection system

Fig. 2 shows the schematic of the single-channel detection system. As already described the first stage is constituted by a CSA. A hysteresis comparator determines the counter observation window.

The two-comparator threshold voltages determine the falling and rising time of the counter clock window.

Fig. 5 shows the CSA output for different values of sensor capacitance and the correspondent comparator windows.

The system measures the absolute values of the cell. A double-channel system can be used

Differential system: capacitive change detection

The system in Fig. 6 is measuring the amount of the change in the capacitance in presence of the target.

Using two channels including two sensors, the reference and the active one and comparing their outputs when the latter is chemically stimulated by an instrumentation amplifier, it is possible to just detect the difference of the capacitive value.

Fully digital differential read-out

Another proposed solution (Fig. 8) acts as a differential read-out EXOR-ing the comparators outputs: the result is directly the difference between the two channels.

In this implementation it is possible to also derive the sign of the differential measurement with a simple finite state machine that observes the rising edges of the comparator outputs. This possibility in the solution with the instrumentation amplifier, described in the previous paragraph, is already imposed by the board routing

Experimental results

A board (Fig. 9) implementing the three solutions described above has been realized and characterized. A micro-controller allows the selection of the three different systems and also to store and process the data.

A perfect correspondence between the simulation results has been achieved by the board characterization [6]. Fig. 10, Fig. 11 show some results from the board test. In Fig. 10 the output of the single-channel read-out is considered: on the top side of the picture the output of the CSA

Conclusion

In this paper, read-out electronics for a capacitive-based DNA sensor have been described and experimental results from a preliminary characterization have been presented. The systems have been proven to be capable of achieving a resolution of 0.01% for the capacitive shift, by using a differential reading of the signals detected by two sensors: a dummy sensor and an active one.

Measurements of the single-channel configuration connected to a sensor prototype confirm the high sensitivity of the

Acknowledgements

This work has been carried out under the framework of the National Project (PRIN 2003) supported from the Ministero dell’Università e della Ricerca Scientifica.

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