ELSEVIER

Biosensors & Bioelectronics Vol. 13. No. 3-4, pp. 451458, 1998 © 1998 Elsevier Science S.A.

All rights reserved. Printed in Great Britain

PII: S0956-5(~3( 97)1~0095-X 0956-5663/981519.00

DNA optical sensor: a rapid method for the detection of DNA

hybridization

Xiao Chen, a Xian-En Zhang, a* Yi -Quan Chai, ~ Wei-Ping Hu, ~ Zhi-Ping Zhang, ~ X iao -Me i Zhang ~ & A n t h o n y E. G. Cass b

aWuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071 PR China bDepartment of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK

(Received 7 October 1996; revised form received 11 August 1997; accepted 12 August 1997)

Abstract: A DNA optical sensor system is proposed based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for rapid detection of DNA hybridization. Bacterial alkaline phosphatase (phoA) gene and Hepatitis B virus (HBV) DNA were used as target DNA. A biotinylated DNA probe was used to capture the target gene onto the streptavidin-coated magnetic beads and a calf intestine alkaline phos- phatase (CAP)-labelled DNA probe was used for subsequent enzymatic chemi- luminescence detection. The detection cycle was less than 30 min, excluding the DNA hybridization time, which was about 100 min. Both the phoA gene and HBV DNA could be detected at picogramme or femtomole level. No response signal was obtained when target DNA did not exist in the sample. Successive sample detection could be made by removing the magnetic field and a washing step. © 1998 Elsevier Science S.A. All rights reserved.

Keywords: biosensor, DNA hybridization, alkaline phosphatase, biotin, streptavi- din, magnetic bead, chemiluminescence, phoA, HBV, flow injection

INTRODUCTION

Nucleic acid hybridization is a basic method in molecular biology and provides new possibilities in various biomedically and biotechnologically oriented fields. Radio-labelled polynucleotide pro- bes have been employed extensively for the detec- tion of complementary nucleic acids by specific hybridization. Unfortunately, radioactive labels are short-lived and require special handling. Most

*To whom correspondence should be addressed. Tel: 0086 27 7641492 Fax: 0086 27 7885422 E-mail: x.zhang@pentium.whiov.ac.cn

importantly, the time required for hybridization of the probes and for autoradiographic detection may be several days. Recently, considerable atten- tion has been given to methods for incorporating non-radioisotopic labels into polynucleotides in order to circumvent the problems inherent in radioactivity. Various techniques have been developed to replace traditional methods using radioactive substances. Biotin, digoxigenin, enzyme, chemiluminophore and fluorescent dyes have all become popular reagents for labelling (Ranki et al., 1983; Urdea et al., 1987; Doom et al., 1994). However, these methods take at least 10 h for detection and require skilled personnel to perform the complicated operations. As a result, a

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growing interest has arisen in DNA detection using biosensor techniques. These methods, termed 'DNA sensors' or 'DNA biosensors', like most other biosensors, feature quick and direct detection.

Earlier DNA sensors may include the piezoe- lectric technique (Fawcett et al., 1988) and sur- face plasmon resonance (SPR) (Evans & Charles, 1990). The former measures a change in fre- quency which results from adsorption of target DNA that hybridized with the DNA probes immobilized on the crystal. This idea was further demonstrated by other investigators ( S u e t al., 1994; Nicolini et al., 1996). The latter measures a change in refractive index of the immediate vicinity of a metal surface when a ligand in solution binds to a ligand receptor immobilized on the metal film. Similar investigations were made recently (Devries et al., 1994; Kruchinin & Vlasov, 1996). Both methods showed their sensi- tivity at picogramme level. But the cost, especially that of SPR, remains to be reduced.

Electrochemical detection of DNA has become of interest recently because of its low cost and reagentless assay. A number of chemical elec- trodes have been used as the working electrodes, such as Basal plane pyrolytic graphite (BPPG) electrodes (Hashimoto et al., 1994), carbon paste electrodes (Millan et al., 1994), hanging mercury drop electrodes (HMDE) (Teijeiro et al., 1993), glassy carbon electrodes (Palanti et al., 1996) and screen-printed electrodes (Wang & Cai, 1996). After modification with complementary DNA fragments or specific molecules, the electrodes were able to detect electrochemically target DNA molecules under certain conditions. The detection limit was at the nanogramme level.

Another alternative is to detect DNA with optical sensors in the combination of either a fluorescence dye system such as Ethidium bro- mide (EB) (Piunno et al., 1995) or acridine (Wu et al., 1996) or an enzyme amplification system (Defillipo & Grayeski, 1991). EB is the most common fluorescent dye for DNA detection in gel electrophoresis. A 20-met ssDNA thymidylic acid was synthesized on the surfaces of derivative quartz optical fibres. The covalently immobilized oligomers were found to hybridize with comp- lementary ssDNA from solution. Hybridization on optical fibres was detected by the use of EB. This method provided a detection limit of 86 ng/ml DNA and a sensitivity of a 200% fluor- escence intensity increase per 100ng/ml of

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cDNA. In the enzyme amplification system, bioti- nylated DNA probes were immobilized onto a membrane and linked to biotinylated glucose oxi- dase by a streptavidin bridge. The GOD-labelled sample was incubated in a solution of glucose. The enzymatically produced H202 was determ- ined using flow injection with a peroxyoxalate chemiluminescence reaction.

Sandwich solution hybridization is a newly developed method for quick DNA hybridization and detection (Ranki et al., 1983; Syvanen et al., 1986). Two sets of DNA probes are exploited. One is labelled with a ligand, e.g. biotin, the other is labelled with a marker such as an isotope, fluorescent dye, semiantigen, or enzyme. These two sets of probes are designed to be complemen- tary to different locations of the target DNA. The hybridization is performed in solution rather than on a membrane, like the conventional method. Since the mobility of the molecules in solution is faster than on a membrane, the hybridization process is speedy. Separation of the hybrid from the reaction solution may be the main problem. Magnetic beads coated with a ligand receptor, e.g, streptavidin, could be a good solution (Huang et al., 1994). The target DNA is captured on to the beads through the binding of ligand and receptor. The complex can thus be separated from the solution by magnetic adsorption. The assay is then performed through the labelled marker reaction, usually an enzymatic reaction. The most frequently used enzymes are glucose oxidase (GOD), horseradish peroxidase (HRP), calf alka- line phosphatase (CAP), etc. Among them CAP may be the most attractive for its high turnover rate and flexibility of signal measurement, either colorimetric or chemiluminescence measurement. Some 1,2-dioxetane derivatives have been developed as substrates of CAP. They can be dephosphorylated by CAP to produce stable inter- mediates that produce a high yield of light output through electron transfer inside the molecule (Beck et al., 1989; Karger et al., 1993). The latest such substrate is CSPD TM (Disodium-3-(4- methoxyspiro ( 1,2-dioxetane-3,2'-(5"- chloro)tricyclodecan)-4-yl)phenyl phosphate), which produces stronger chemiluminescence (Martin & Bronstein, 1994; Bronstein et al., 1994; Kricka et al., 1994) and is commercially avail- able.

The purpose of this study is to develop a sensitive, easier to operate and reusable sensor system for rapid detection of DNA hybridization.

Biosensors & Bioelectronics DNA optical sensor

It combines several techniques mentioned above: non-radioactive probes, sandwich solution hybridization, magnetic bead capture, flow injec- tion analysis and chemiluminescence. Fig. 1 shows the principle of the hybridization. Only in the presence of the target DNA was the signal observed in the detection.

MATERIALS AND METHODS

Reagents

CSPD TM was obtained from Boehringer Mannheim AG (Switzerland). Streptavidin Mag- neSphere Paramagnetic Particles, CAP (EC 3.1.3.1., 1 U/ml) and the Wizard TM Minipreps DNA Purification System were purchased from Promega, Madison. Biotin hydrazide, p-benzo- quine and polyethyleneimine (PEI) were from Sigma Chemical Co. (St Louis, MO) Vent DNA Polymerase and molecular weight markers were from New England Biolabs, UK. Low melting- point agarose was purchased from BRL (Gaithersburg, MD). Plasmid pEK48, which car- ries the p h o A gene, and E. coli SM547, which has a deletion of the p h o A gene, were donated by Dr R. Kantrowitz (Boston College, MA).

DNA preparation and hybridization

The following procedure is for the preparation of p h o A gene fragments. E. coli SM547 was grown in LB broth. After transformation with pEK48 containing p h o A gene, E. coli SM547 was grown again in LB broth supplemented with ampicillin (100/~g/ml). Transformants containing the plas- mids were identified according to their sensitivity to ampicillin and screened by analysing the size of the plasmid DNA by agarose gel electro- phoresis. A Wizard TM kit was used according to the manufacturer's instructions for plasmid prep- aration and purification. The PCR procedure was as follows: a 5 min pre-incubation at 94°C, fol- lowed by 30 cycles of 1 min at 94°C, 2 min at 50°C and 3 min at 72°C. After the last cycle, an extra incubation for 30 min at 72°C was carried out. The reaction mixture of 100/xl contained 1 /xl (1 /xg) pEK48 template, 2/xl (100pmol) primer, 1 U Vent DNA polymerase, 100/xmol/1 deoxynucleotide triphosphate in a buffer contain- ing 10 mmol/1 KC1, 10 mmol/1 (NH4)2804, 20mmol/1 Tris-HC1 (pH8-8, 25°C), 2mmol/1 MgSO4 and 0.1% Triton X-100. The reaction mixture was covered with a layer of mineral oil. The product of the PCR reaction was analysed by 1.7% agarose gel electrophoresis in the pres-

Target ssDNA (3 ~ridiz~

CAP-labeled probe Biotinylated probe

Streptavidin coated magnetic bead

In situ enzymetic reaction & signal processing

Fig. l. Principle o f sandwich solution DNA hybridization and magnetic bead binding. The hybridization is performed in solution. The target ssDNA is hybridized with two kinds of probes, a CAP-labelled probe and a biotinylated probe. The hybrids are then bound to the streptavidin-coated magnetic beads through biotin-streptavidin binding reaction. The whole process takes about 130 min. After washing to elute unbound DNA the sandwich beads are

ready for measurement.

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Xiao Chen et al. Biosensors & Bioelectronics

ence of ethidium bromide using long-wave ultra- violet light transillumination. It was purified further from a low melting point agarose gel using the Wizard TM kit.

Hepatitis B virus (HBV) DNA hybrids were prepared according to Wu & Cai (1993). Two sets of DNA probes were used in hybridization. One was a biotinylated probe which was prepared using the method described by Reisfeld et al. (1987), which is based on the interaction of biotin hydrazide with unpaired cytosine residues of ssDNA. The interaction is catalysed by sodium bisulphite with an optimum at a buffered pH of about 4.5. Another one was labelled with CAP, using the method described by Renz & Jurz (1984). CAP was coupled with PEI using p- benzoquine as a cross-linking reagent. The modi- fied phosphatase was covalently linked to single stranded DNA using glutaraldehyde. The sequences of both probes were complementary to different regions of the target DNA.

Various amounts of denatured target DNA (phoA or HBV) were diluted to the final concen- tration. Two sets of DNA probes were added in a five-fold molar excess over the maximum target fragment concentration. Hybridizations were car- ried out in plastic Eppendorf tubes under the following conditions: 10/xl of 20 x SSC, 5/xl of 100 x Denghardt 's with shaking for 100 min at 37°C in a final volume of 20/xl. A 'no target' control was run with each series. Paramagnetic streptavidin-coated particles were prewashed twice with 0.5 x SSC. Fifteen microlitres of beads (0.15 mg) were added to each sample (hybridization products) and incubated for 30 rain at room temperature. Then the mixture was washed twice with 0.15 mol/1 LiC1, 0.1% SDS, 0.01 mol/1 Tris-HC1 (pH 3.0), and twice with 0.5 x SSC.

Measurement procedure

The washed mixture was mixed thoroughly and the suspension was pumped into the flow line of the working system of the DNA biosensor as depicted in Fig. 2. When passing the measuring cell in the dark box, beads with their captured DNA were trapped at the bottom of the cell by using a magnetic separation stand which causes them to adhere to the cell. The time to stop flow is dependent on the flow rate, and distance and was optimized to ensure that the bead-capture mixture could be thoroughly incubated in the

chemiluminescent buffer (0-1 mol/1 DEA, 1 retool/1 MgC12, 0.02% NAN3, pH 10-1) contain- ing CSPD. Light output was obtained on a photo- dynamometer. Output could also be given as a full integral of the light produced during a certain time with assistance of the computer. After measurement, the magnet was removed and beads were ejected by washing with large quantities of suitable buffer. A second sample could then be detected right away.

RESULTS

Plasmid pEK48 DNA (5-7 kb) was purified using the Wizard TM kit. Four kinds of primers were prepared and three sets of PCR reactions were run, all of which used the pEK48 DNA as the template. Three kinds of PCR products, fragments AB (982 bp), BC (346 bp) and AD (196 bp), were obtained. Fragment AB (phoA gene) was used as the target DNA. The AD and BC fragments were labelled with biotin and CAP, respectively.

The pH dependence of CAP-activated chemi- luminescence from CSPD was tested with 5/xl of 10 -14 mol/1 of free CAP and 10/xl of 3 mmol/l of CSPD solution as depicted in Fig. 3. The optimum pH in the assay was in between pH 10.1 and 10-2. The subsequent experiments were car- ried out at pH 10.1. Various concentrations of free CAP were used to investigate the detection limit for CAP with CSPD (Fig. 4). Intensity of chemiluminescence was directly related to the concentration of CAP ranging from 10 |7 to l0 12 tool. This means that if one DNA probe were labelled with one enzyme molecule, the theoretical detection limit would be 10 lV mol DNA.

Various amounts of AB fragments of the phoA gene were detected using the DNA optical sensor. After the sandwich solution hybridization and the bead capture, 20/xl of chemiluminescent buffer containing 5 mmol/1 of CSPD was pumped into the cell. After 10 min incubation at room tem- perature, the luminescence intensity was read for 20 min (Fig. 5). Light output increased with the amount of target DNA in a sample but did not show a linear relationship. The response signals were picked up manually every 10 s during a 20 min measurement course and were integrated with the assistance of a computer using Fox pro software. The resultant data became more read- able (Fig. 6). Much higher sensitivity was then

454

Biosensors & Bioelectronics DNA optical sensor

Recorder

CSPD Darl

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.................... llllllltl

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Buffer ~ Waste • [ _ _ _ _ •

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Paramagnetic SA-coated Beads with entrapfed DNA Sample

Fig. 2. Schematic diagram of the working system of the DNA optical sensor. The suspension of sandwich beads is pumped into the flow line of the working system. Beads with their captured DNA are trapped at the bottom of the measuring cell by a magnetic separation bar that stands outside the cell. The time to stop flow is optimized to ensure that the bead-capture mixture can be thoroughly incubated in the chemiluminescent buffer (0.1 mol/l DEA, 1 mmol/l MgCl2, 0.02% NAN3, pH 10.1) containing CSPD. Light output is obtained on a photo-dynamometer and recorded by a chart recorder. Output could also be given as a full integral of the light produced during a certain time with the assistance of a computer. After measurement, the magnet is removed and beads are washed out,

allowing the subsequent measurement.

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at 4 min as a 5 s integral.

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455

Xiao Chen et al. Biosensors & Bioelectronics

1 . 0 , , , ~ • , • , . ~ i . , , , • , • ,

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Fig. 5. Chemiluminescence signal accumulation during incubation of CSPD with hybridization mixture at dif- ferent phoA concentrations. Analytes were incubated with 20 txl of reaction buffer (5mmol/l CSPD in 0.1 mol/l DEA buffer) at room temperature for 10 mitt.

The luminescence intensi~ was integrated for 5 s.

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Fig. 6. Signal integration during incubation of CSPD with hybridization mixture at different phoA concen- trations. The data were derived from Fig. 5 and pro- cessed by computer. A much higher sensitivity was

obtained in this way.

obtained. For example, between a control sample and 10 pg of p h o A DNA, differences of response signals were 0.2 × 10 -8 mW at the twentieth minute of incubation time and 0-28 × 10 -5 mW at 30 min, while difference of integrated data was 3 x 10 -8 and 18.6 × 10 8 mW.

Twenty and 200pg of HBV DNA (0.3 kb) were detected using the same procedure but with different probes. The intensity was again related to the amount of HBV DNA (Fig. 7).

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Fig. 7. Chemiluminescence signal accumulation during incubation of CSPD with hybridization mixture at dif- ferent HBV DNA (0.3 kb) concentrations. The measure-

ment process was the same as in Fig. 5.

DISCUSSION

The method described in this communication employs a number of new techniques and is thus advantageous in several aspects. Firstly, DNA hybridization in solution is much faster than on a membrane; it minimizes the time course of the detection. Secondly, by using magnetic beads separation of DNA hybrids occurs in a single closed vesse l - -no columns, no pumps, no centri- fuges, no aerosols. Furthermore, the flow injection manifold and the on/off magnetic field enable all DNA hybrids to be captured inside the measuring cell and washed out after the reaction, resulting in maximum signal generation and avoiding regeneration of the sensor tip. Regeneration of the sensor tip is usually one of the main problems of affinity sensors. Finally, CAP-CSPD is a highly efficient amplification system. In combi- nation with data integration the detection limit for the p h o A gene fragment and HBV gene frag- ment is down to picogrammes, or close to 10-]Tmol, a level that is comparable to pre- vious reports.

As for the sensitivity of the DNA sensors, there is sometimes confusion. Some authors use the weight unit, others use molar units. The mol- ecular weights of target DNA may vary over a wide range, from thousands to hundred thousands or more. Since one molar target DNA molecules can bind no more than one molar market labelled probe, suppose the detection limit is 1 0 - ] 7 mol; if the molecular weight of a target DNA is 105, the detection limit may be 10 ~5 g, or if the

Biosensors & Bioelectronics

molecular weight is 104 , the detection limit may be 10-16g or so. So it is more reasonable to use molar units in the case of DNA sensors.

As shown in Fig. 6, the integration data (accumulated signal) increases obviously at the later stage of the reaction. This is because the output at this stage is higher and stable. The longer the time lasts the higher the ratio of signal- to-noise will be. There is no doubt that the sensitivity of the DNA sensor can be increased further by one or two orders by a data integration method with a built-in computer.

The results do not show a linear response, either in free CAP reaction or in DNA detection, or the linear response is limited to a low concen- tration range. It may be an inherent attribute of the optical sensor, which showed a similar phenomenon in our previous study on determi- nation of glucose. However, detection of DNA hybridization is a qualitative analysis, the answer is 'negative' or 'positive'. A quantitative result is usually not necessary in this sense.

One additional advantage of the approach described here is the potential to conduct multiple assays for different analytes simultaneously. In one format, by changing the enzyme label and the labelling-probe sequences for a new analyte, it should prove possible to detect different ana- lytes in the same sample. Alternatively, by syn- thesizing different analyte-dependent complemen- tary capture probes whose sequences differ from one analyte to another, it should be possible to perform several different assays simultaneously with the same enzyme label.

ACKNOWLEDGEMENTS

This research was supported by the Chinese National Natural Science Foundation and Chinese Academy of Sciences-Royal Society joint project. The authors also gratefully acknowledge the sup- port of the K. C. Wong Education Foundation, Hong Kong.

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