ARTICLE IN PRESS

Microelectronics Journal 40 (2009) 1345–1349

Contents lists available at ScienceDirect

Microelectronics Journal

0026-26

doi:10.1

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

Smart sensors for fast biological analysis

Massimo Lanzoni �, Claudio Stagni, Bruno Ricco

DEIS, University of Bologna, Viale Risorgimento 2, 40136 Bologna, Italy

a r t i c l e i n f o

Article history:

Received 31 March 2008

Accepted 5 August 2008Available online 5 October 2008

Keywords:

DNA

Instrumentation

EEPROM

92/$ - see front matter & 2008 Elsevier Ltd. A

016/j.mejo.2008.08.001

esponding author. Tel.: +39 051 2093078; fax

ail address: massimo.lanzoni@unibo.it (M. La

a b s t r a c t

This paper presents an updated overview of present works concerning the realization of biological

sensors based on electronic devices. In particular, DNA sensors will be described and their main

characteristics will be analyzed. As an example, a new sensor for DNA detection and analysis will be

described in detail. The proposed approach is based on the use of non-volatile memories and does not

require sample treatment nor sensor surface functionalization. Moreover, fabrication technology is

widely compatible with the standard CMOS process. Experimental results show that the sensor has

sufficient accuracy and sensitivity that can be further improved by suitable device engineering.

& 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Increasing research on electronic devices able to perform fastand accurate biological analysis is led not only by industrialinterests in clinical applications but also by antiterrorismprograms.

In recent years a variety of devices have been developed,thanks to new silicon technologies such as micromachining andmicrofluidics, allowing dramatic scaling of equipments usuallybulky, difficult to use and requiring large amount of material forthe analysis.

Medicine, biology, food and ambient analysis require increas-ing DNA sequencing and this motivated a large development ofnew systems capable of answering such demand.

All the proposed devices are based on the effects of possiblehybridization between the single-stranded DNA fragments to beanalyzed (hereafter referred to as ‘‘targets’’) and known to captureoligonucleotide sequences (called ‘‘probes’’ or ‘‘receptors’’),immobilized on a substrate by means of suitable surfacebiological functionalization procedures (hereafter referred to as‘‘bio-functionalization’’).

In recent years, microfabricated devices, normally called DNAmicroarrays [1,2], have been developed for this type of analysis.Microarrays offer significant advantages compared with conven-tional laboratory techniques, in that they allow large parallelism(i.e. a large number of tests can be performed at the same time)and are relatively simple to use in a laboratory. Furthermore, verylittle amounts of sample material and bio-chemical reagents arerequired.

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nzoni).

So far, in these devices DNA hybridization is detected by meansof optical marker-molecules previously attached to the DNAtargets. At present, DNA chips based on optical detection of thehybridization of labeled DNA [3] and capable of testing hundredsof thousand different probes in parallel are technologically maturedevices already in the market. The targets are labeled withchromophores molecules and deposited on the array surface.Successively, all these materials are removed and only the targetsthat have hybridized with complementary probes remainimmobilized on their specific sensing sites. Finally, the device isobserved optically to localize the chromophores, revealing wherehybridization has taken place. Often, the resulting image is notimmediately recognizable, because hybridization can involvemany adjacent sites resulting in a complex optical pattern to beresolved by means of adequate image processing. This technologypresents two main drawbacks: cost of the instrument to detectand resolve the optical signal; necessity of a labeling step, withthe need of additional reagents and the possibility of samplepollution. This method, however, has several significant draw-backs, since: (a) it requires preventive target manipulation tointroduce the optical markers; (b) requires expensive instrumen-tation for optical reading; (c) does not allow real-time detection;(d) can be limited in sensitivity by the non-homogeneity ofmarker distribution. Therefore, alternative approaches are beingstudied and, in this context, new label-free approaches have beenproposed.

To eliminate the former problem, methods based on thegeneration of electrical signals upon hybridization have beendeveloped. However, some of these techniques are not label free,as they use mediator elements to generate the electrical signals[4–7]. Moreover, some innovative microarrays employ electro-chemical labels resulting in an electrical current through sensorelectrodes during readout in case of hybridization events [8–10].

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Fig. 1. Experimental setup.

M. Lanzoni et al. / Microelectronics Journal 40 (2009) 1345–13491346

Naturally, label-free approaches featuring fully electrical read-ing techniques represent in principle the simplest, most direct,hence also best solution.

Recently, a number of approaches have been proposed to thispurpose based on mass changes [11–15] or electrical propertiesof electrode/solution interfaces induced by DNA hybridization[16–19]. In particular, a simple label-free approach based onelectrode/solution interface has been investigated. This pheno-menon has been studied extensively in the past, using aconventional electrochemical three electrodes system, by apply-ing a 50 mV potentiostatic voltage step and measuring theelectrode current [20]. In [21], bio-functionalized gold electrodeswere characterized with impedance spectroscopy. Moreover, asimplified setup based on two gold electrodes and capacitancemeasurements has been proposed exploiting only microfabricatedelectrodes and standard instrument [22]. Moreover, an activeCMOS sensor chip is required in applications where a largenumber of analyses have to be performed in parallel. Then, notonly the sensitivity of the single sensor site is increased, but alsothe electrical interconnect to the outside world is reduced: twoworks have shown results in this direction [23]. Nevertheless,problems related to reproducibility and stability of measurementsindicates that application out of research laboratories will stillneed much efforts.

Optical detectors have been proposed too. In particular, in thistype of sensors, some optical properties are modified by DNAhybridization with targets placed on some kind of optical surface.As a result, intensity [24] or frequency response [25,26] of thestructure can be changed depending on the sensor structure andcharacteristics.

Finally, floating gate (FG) CMOS devices have been proposed ashybridization detectors [27]. In this work, DNA hybridization on apart of the FG of a CMOS non-volatile memory device can inducea threshold shift due to the electric charge associated to theDNA molecules that can vary in number and mass by effect ofthe reaction with targets fixed on the exposed surface of thepolysilicon FG.

In the following sections, we describe a new DNA hybridizationsensor based on UV detector devices that presents significantdifferences with respect to those presented before. In particular,DNA selective hybridization takes place on a quartz slide separatefrom the sensor. In addition, FG devices are used as UV sensors.

2. DNA hybridization system working principle

At system level, our approach envisages an array of ‘‘sensing’’sites, each consisting of a bio-layer of specific probes, positionedbetween an external UV source and a UV detector. The bio-layer isconventional, in that it is obtained by means of DNA probes,aligned with the underlying sensor and working as ‘‘selectiveglue’’ with respect to the DNA target molecules.

Fig. 1 gives a schematic representation of the experimentalsingle-site detection system. As can be seen, DNA probes are notattached to the sensor but to a quartz support optically aligned tothe UV detector.

In operation, all sites are simultaneously exposed to the targetmolecules, that will selectively hybridize only with complemen-tary probes. Since DNA has a significant UV absorption, the siteswhere hybridization has taken place will present different UVabsorption with respect to those where the same amount of DNAis in the non-hybridized state. The radiation transmitted throughsuch sites will be smaller in the first case, due to masking effectof the hybridized DNA. As already explained, the recognitionof this difference implies that of complementary molecules.Among the advantages of this new approach the most relevant

are: (a) hybridization area is separate from the UV detector,thus does not need to be cleaned or disposed after use; (b) nopretreatment of the biological material to be analyzed is required,in particular no addition of optical markers is needed; (c) UVdetectors presented in this work are fabricated with the standardCMOS technology and can be dimensionally scaled to lowdimensions for microarray fabrication.

In this work, we focus on an implementation aimed at high-performance devices, exploiting all the advantages of siliconintegration. In particular, both the UV sensors and the electronicsneeded to address and read the individual sites could beintegrated on the same chip and the resulting devices would beextremely compact, fast and suitable for high-density arrays ofsensing sites.

In particular, this work investigates the use of NV memory cellsas UV sensors for DNA detection/recognition and describes theexperimental characterization of single-poly EEPROM transistors,shown to be particularly suitable for the purpose.

Because of the original solution adopted for the UV sensors,this paper represents a significant advancement in the state-of-the-art in the field of DNA sensors/detectors, with possibleextension to protein analysis.

3. CMOS FG devices as UV detectors

The key point of this work is the use of NV memory cells as UVsensors. As known, these cells can be erased by means of UVradiation, particularly if designed so that their FG can be directlyexposed to the incoming radiation.

Since during erasing the cell threshold voltage shift(DVTH ¼ VTH0–VTH, where VTH and VTH0 denote the present andinitial values of the threshold voltage, respectively) increases withthe UV dose, NV cells represent almost ideal UV dosimeters thatcan be exploited for the purpose of this work.

Therefore, the DNA chip envisaged in this work features anarray of suitable NV memory cells, all initially programmed atthe same threshold voltage VTH0 (bVTHN where VTHN is the cellthreshold voltage with no charge on the FG). Each cell (or group ofcells) is located below a specific bio-functionalized layer. Afterexposure to the target DNA the whole array is exposed to UVradiation for a fixed time. Consequently, all cells are partiallyerased, but those covered by hybridized targets receive lessradiation, hence exhibit a final higher value of VTH. (i.e. a smallerDVTH), compared with those where non-DNA binding hasoccurred. Of course, all the electronics needed to individuallyselect and read the cells in the array are essentially the same as in(multi-level) memories, hence can be integrated on the same chipas the cells.

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M. Lanzoni et al. / Microelectronics Journal 40 (2009) 1345–1349 1347

For the purpose of this work ‘‘single-poly EEPROM devices’’ aresuitable for this application. In fact, in this technology, the controlgate (CG) is realized by means of an n+ diffusion under the FG,that extends outside the channel area (Fig. 2). This feature hassome very important benefits, namely: (a) the FG is completelyexposed to UV radiation (i.e. no ‘‘masking’’ CG is present); (2) theFG area is larger than normal (thus more UV radiation canbe collected); (3) the cell can be easily fabricated by CMOStechnology.

In order to characterize system effectiveness, liquid DNAsolutions in quartz containers have been ‘‘superimposed’’ tomemory cells. In this way, DNA concentrations can be controlledwith great accuracy. The DNA ‘‘sensing site’’ used in this work isshown in Fig. 2.

A quartz container with the DNA targets is placed between aUV lamp and the NV memory cell used as a dosimeter for theimpinging UV radiation. A mechanical shutter allows to switch onand off the incident UV radiation.

As for recognition of specific DNA sequences, the experimentsof this work exploit the so-called hypochromic effect, namely thefact that for the same number of DNA bases (nucleotides), the UVabsorption of double-stranded molecules is smaller (by about20%) than that of the single-stranded form. Of course, for thepurpose of this work a major question concerns the sensitivityof NV cells used as UV detectors and, from this point of view,a suitable device has to be used.

Fig. 2. DNA detection principle.

DNA - 5400 nM single stranded

DNA - 3600 nM single stranded

DNA - 1950 nM single stranded

DNA - 900 nM single stranded

Buffer solution TE 1x (1.5 ml)

ΔVth

[V]

0

0.4

0.8

1.2

1.6

2

Dos

0 50 100

Fig. 3. Variation of EEPROM cell threshold voltage shift as a function of the

4. Experimental setup

In the experimental setup used in this work the source of UVradiation is a xenon lamp characterized by a spectral distributionwith high emission values in the range of interest (250–270 nm).The lamp filament is controlled by a special circuit whichmaintains the supplied power constant. However, since this doesnot guarantee radiation stability over long time periods, a specificcircuit (based on commercial UV sensitive photodiode) has beenused to measure the instantaneous radiation and compute thetotal dose.

The sample data are postprocessed by a LabVIEW program inorder to evaluate the radiation dose of the experiments. The sameprogram controls the shutter and switches off the radiation oncethe dose has reached a desired value.

The EEPROM VTH is measured, when the light source isdisabled, by means of a HP4156 semiconductor parameteranalyzer (SPA) driven by the control PC. Resolution in VTH

measurements is 1 mV.For each DNA concentration, measurement is repeated 5 times

to evaluate the standard deviation, hence the measurementreliability. Resulting data present very low standard deviation,thus no error bars have been plotted.

5. Experimental results

The results described in this section are shown as a function ofUV dose (a parameter equivalent to, but more appropriate thanexposure time).

Fig. 3 shows the typical behavior of DVTH as a function of theUV dose and, as can be seen, a difference is measured for differentvalues of single-stranded DNA concentration. Similar results wereobtained with double-stranded DNA.

Since the significant parameter to be analyzed is the differencebetween the buffer solution and that containing a certainconcentration of DNA, we introduce a new parameter denotedas under erasure (U.E.) defined as

U:E: ¼ DVTH_BUFFER � DVTH_DNA, (1)

where DVTH_BUFFER and DVTH_DNA represent DVTH measured inthe case where only the buffer or the buffer containing DNA isinterposed between the UV source and EEPROM cells, respectively(in this definition, of course, DVTH_BUFFER merely represents aconvenient common reference).

e [A.U]

150 200 250

UV dose for the case of single-stranded (i.e. non-hybridized) molecules.

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U.E

. [m

V]

Base concentration [nM]0 1000 2000 3000 4000 5000 6000

ΔVth for DNA double stranded

ΔVth for DNA single stranded200

160

120

80

40

0

Fig. 4. U.E. for the case of hybridized and non-hybridized DNA, lower and upper

curves, respectively.

M. Lanzoni et al. / Microelectronics Journal 40 (2009) 1345–13491348

The most relevant results obtained are illustrated in Fig. 4,where the experimental points are drawn in such a way that eachvalue of concentration on the x-axis corresponds to the samenumber of single-stranded DNA bases (i.e. the elements actuallyabsorbing UV radiation), so that the difference among the variouscurves is due only to whether or not the DNA is in the bound(i.e. hybridized) state. In particular, the points indicated withsquares and diamonds represent the case where the same number(N) of DNA single strands are presented in double- and single-stranded form, respectively.

As can be seen, the measurements allow to easily recognize thecase of hybridization, that is the key for the DNA sensor envisagedin this work.

6. Discussion

The results presented before clearly indicate that a DNAmicroarray based on the approach suggested in this work andrealized in a single chip with standard CMOS technology seemspossible, since (at least) single-poly EEPROM cells represent asuitable UV detector.

The results of Fig. 4 indicate that double- and single-strandedmolecules in 1 cm path length can be distinguished startingfrom a minimum concentration of 1.5mM bases (1.5 nmol/cm2).In the case of microfabricated devices, the height of the containershould, of course, be reduced, for instance down to 100mm,hence for the same number of bases along the optical paththe DNA concentration should be increased to 15mmol/cm2.Thus, in a microwell containing for example 106mm3 ofsolution, the absolute number of bases needed to distinguishbetween hybridized and non-hybridized samples is 1.5 nmol,a quantity definitely compatible with that usually employedfor analysis.

7. Conclusions

This paper provides a significant contribution toward therealization of innovative DNA sensors fabricated in a single chipwith standard CMOS technology and based on UV absorption ofDNA molecules (not yet used for microfabricated devices).

At this regard, the paper proposes to use non-volatileEEPROM cells that represent almost ideal UV dosimeters. Morespecifically, the suggestion is made to use single-poly memory

cells that can be fabricated with the standard CMOS technology,and can be easily integrated with all the required electronics(for addressing and reading) and present a number of significantadvantages.

The experimental results described in the paper indicatethat such devices have adequate sensitivity to distinguishbetween cases where DNA (unknown) target molecules hybri-dize with (known) probe sequences and those where this isnot the case, clearly the essential point for the use envisaged inthis work.

Acknowledgment

The authors are grateful to ST-Microelectronics (Milan, Italy)for providing the devices used in this work.

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