Short communication Label-free amplified …

1 Biosensors and Bioelectronics 20 (2004) 658 662 Short communicationLabel- free amplified bioaffinity detection using terahertz wave technologyAbdellah Menikha,b, , Samuel P. Mickana,c, Haibo Liua, Robert MacCollb, ZhangaaCenter for terahertz Research, Department of Physics, Applied Physics&Astronomy, Rensselaer Polytechnic Institute,110 8th Street, Troy, NY 12180, USAbWadsworth Center, Box 509, Albany, NY 12201, USAcCenter for Biomedical Engineering, Department of Electrical Engineering, University of Adelaide, Adelaide, SA 5005, AustraliaReceived 10 December 2003; received in revised form 28 February 2004; accepted 4 March 2004 Available online 21 April 2004 AbstractA new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidinmolecules.

2 Amplified detection of avidin biotin binding is obtained on supported membranes composed of biotin layers on quartz surface,which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to theoctadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin andbiotin avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was ableto detect less than ng/cm2avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry andreflectometry techniques.

3 Further improvement is underway using highly refractive beads together with appropriate surface chemistry. Thisnewly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand analyteaffinity binding. 2004 Elsevier All rights :Biosensor; Optics; Avidin; Biotin1. IntroductionIn the panoply of biosensor technology, many trans-ducers have been developed and used successfully fordetection purposes. In the UV-Vis optical region, detectionof ligand analyte binding is mainly accomplished by theapplication of either surface plasmon resonance biosensor(SPR) or fluorescence spectroscopy (Smith et al.)

4 , 1981;Mayo and Hallock, 1989). These methods have registeredenormous success and are now being used routinely in ana-lytical laboratories. However, to the best of our knowledge,the number of biosensors operating in the far infrared regionis either scarce or non-existent. The techniques that havebeen recognized as potentially useful tools for monitoringon off binding in the far infrared region are attenuated totalreflection Fourier transform infrared (ATR-FTIR) and Ra-man spectroscopy (Brown et al., 1972; Regan et al., 1996).Unfortunately, the complications associated with cryogenicdetectors in FTIR, and Raleigh lines in Raman spectroscopy Corresponding author.

5 Tel.:+1-518-2763079; fax:+ (A. Menikh).are among the anomalies that hampered these techniquesfrom being widely used. Recently, a new detection methodbased on terahertz differential time-domain spectroscopy(THz-DTDS) has been proposed and used successfully todetect minute amounts of Label-free antigen antibody bind-ing, lipid protein interactions, DNA hybridization and drugdiscovery (Nagel et al., 2002a; Menikh et al., 2002).In the ever-advancing field of biosensing systems there isalways a need for methods to increase system sensitivity tosignals that indicate binding of one biological molecule toanother. There is also a desire for flexibility in the types ofligands that mimic phenomena occurring in nature.

6 To sat-isfy these criteria, we have developed a novel biochemicaltechnique, based on small agarose beads, which can offerone such solution owing to some of their unique features:Small agarose beads provide a large outer surface area onwhich target molecules can be attached. Spherical shapedbeads provide a very flexible, cell membranes-like environ-ment. Therefore, by employing a secondary interaction withsmall agarose beads, conjugated with an analyte, the THz op-tical difference signal between a ligand and a ligand analyteaffinity bound component can be $ see front matter 2004 Elsevier All rights Menikh et al.

7 / Biosensors and Bioelectronics 20 (2004) 658 662659 Avidin and biotin molecules have been chosen in thisstudy due to their unique binding properties. Avidin is aprotein, which is comprised of four identical subunits, eachbinding one biotin molecule. The association affinity be-tween avidin and biotin is so high (Ka=1015mol 1) thatthe formation of this complex can be regarded as nearlyirreversible, on a scale comparable to a covalent bonding(Mickan et al., 2002). The high affinity binding of this sys-tem has found many applications, including affinity chro-matography, attaching antibodies to solid surfaces, precipi-tating liposomes, and targeting cells with liposomes (Green,1975; Cuatrecasas and Wilchek, 1968).

8 Importantly, it hasbeen shown that biotin can be adsorbed on hydrophobic sur-faces without losing its specificity towards avidin, whichmakes possible the study of avidin biotin interactions onsupported lipid aim of this paper is to demonstrate the capabilityof biochemical means to amplify the optical terahertz sig-nal from a previously affinity-bound compound in a lipidmembrane-like environment. This chemical amplificationmethodology is simple, non-invasive, inexpensive, anddoes not require hardware modification as in Materials and ChemicalsThe following chemicals were obtained from Sigma ( , MO), and used as obtained without further purifica-tion.

9 Octadecanol, bovine serum albumin (BSA), biotin, andagarose beads conjugated to avidin. Digalactosyldiacylglyc-Fig. 1. (a) Octadecanol self-assembled modified quartz crystal surface; (b) biotin monolayer on a quartz surface modified octadecanol; and (c) biotin avidinbinding in lipid membrane like (DGDG) from spinach leaves was obtained from LipidProduct (South Nufield, UK). Quartz microscope slides(25 mm 25 mm) were obtained from Electron MicroscopySciences (Washington, PA), and tested for thickness homo-geneity before use. All reagents were HPLC grade and usedas received. Aqueous solutions were prepared in doublydistilled de-ionized Sample preparationPrior to deposition, the quartz slides were cleaned in50% hot nitric acid for 1 h, and then rinsed thoroughly indoubly distilled water.

10 The quartz slides were further sub-jected to 1 mg/ml octadecanol solution for half an drying and washing with doubly distilled water, thesample was dipped in mg/ml biotin dissolved in chlo-roform/methanol (5:1) and allowed to incubate for 45 spontaneous organization of the biotin lipid into oc-tadecanol self-assembled bilayers guaranteed a certain levelof stability for the octadecanol-biotin complexes. To elimi-nate the effect of non-specific interactions, the quartz slideswere further incubated in 1% bovine serum albumin forhalf an hour, dried and washed several times with a phos-phate buffer solution (PBS).