WO2007074348A2

WO2007074348A2 - Multianalyte biosensors based on monolithic optoelectronic transducers

Info

Publication number: WO2007074348A2
Application number: PCT/GR2006/000069
Authority: WO
Inventors: Sotirios Kakabakos; Konstantinos Misiakos
Original assignee: National Center For Scientific Research (Ncsr) Demokritos
Priority date: 2005-12-27
Filing date: 2006-12-27
Publication date: 2007-07-05
Also published as: WO2007074348A3; GR1005589B; GR20050100623A

Abstract

Monolithic optical biosensors for sensing biomolecules are disclosed. In one aspect, the biosensors comprising a plurality of light-emitting elements and optical waveguides, each waveguide optically coupled to a light-emitting element; and an optical detector, wherein the optical biosensor is adapted to detect an analyte by binding of the analyte on a surface of one or more of the waveguides and thereby changing the optical coupling between the optical detector and one or more of the light emitting elements. In another aspect, a biosensor comprises a light emitting element, an optical waveguide optically coupled to the light emitting element and to an optical detector, the optical waveguide having a plurality of surface features adapted to effect a change in optical coupling between the light emitting element and the optical detector when biomolecules to be sensed are provided over the waveguide.

Description

Multianalyte Biosensors Based on Monolithic Optoelectronic

Transducers

Field of the Invention The invention relates to the field of optical multianalyte biosensors with multiplexed output signal.

State of the Art

The field of portable bio-analytical devices performing diagnostic clinical or environmental assays is rapidly expanding because of social trends and economic reasons. Personalized health care and monitoring of environmental hazards for the general public have been accepted as life quality standards in industrialized countries. Reduced assay costs and home diagnostics present a long waited alternative to centralized clinical laboratories and extended hospitalization times. Such devices are now possible due to the latest developments in micromachining based microsystems technology and co-integration of silicon with a number of devices and material structures ranging from bio-chemical add-on layers to mechanical parts and electromagnetic components.

It is especially desirable that analytical microsystems be fabricated monolithically. So far, analytical microsystems are assembled by the hybrid integration of a components set. However, hybrid integration is costly, it complicates packaging and besides, it becomes impractical in multi-functional multi-target detection microsystems. This represents a problem especially for optical Microsystems, where precise alignment of the light sources to the transducer and detector subsystem is of critical importance and determines performance.

On the other hand, the use of silicon based micro-technology could yield inexpensive and monolithically integrated Microsystems as it is disclosed in WO 03046527 and in K. Misiakos et al. Anal. Chem. 76, 1366-1373, 2004.

The present invention presents a solution to this need through the simultaneous fabrication of a large number of microsystem chips by the processing of large diameter silicon wafers. The present invention addresses some key issues in order for such bioanalytical Microsystems to be able to compete even more effectively with traditional analytical techniques. Such key issues are

- Multianalyte potential, without increasing the system requirements on readout electronics and on micromechanical-microfluidics architecture, as well as - detection with no additional reagents (label-free detection).

At the same time the invention advantageously employs optical detection to take advantage of the galvanic isolation of the transducer from the excitation and detection electronics.

Such a monolithic optical bioanalytical device is disclosed in this patent, which device advantageously uses the benefits of optical detection in a self-aligned monolithic silicon optical micro structure featuring single node readout for a large number of optocouplers. The invention further enables label-free detection based on the use of noble metal nanoparticles or periodic-photonic formations on the waveguide.

With regard to prior art documents such as WO03046527 and K. Misiakos et al. Anal. Chem. 76, 1366-1373, 2004, the invention embodies two major innovations. More specifically, the monolithic optoelectronic transducer presented in WO03046527 (Al) and in K. Misiakos et al. Anal. Chem. 76, 1366-1373, 2004, consists of an array of silicon optocouplers made of silicon Light Emitting Diodes (LEDs), silicon nitride waveguides and silicon p-n junction detectors. The silicon LEDs are avalanche diodes biased beyond their breakdown voltage. The silicon nitride fibers are self aligned with respect to the silicon nitride waveguides by employing a silicon dioxide spacer and implantation of the avalanche diode emitter through the silicon nitride layer. The silicon nitride waveguide optically connects the light emitter to the detector so that a monolithic silicon-based optocoupler is built. To transform the optocoupler into a biosensor the waveguide is functionalized by immobilizing capture biomolecules that later recognize the analyte biomolecules in the sample and bind to them. At the end of the bioanalytical process photon absorbing labels like chromophore groups or metal nanoparticles are employed in order to follow the binding reaction of the biomolecules immobilized on the waveguide with the analyte in the sample. The device described in the above mentioned references employs a number of independent optocouplers where each optocoupler has its own detector.

The major problems encountered with this technology are the following: a) complicated control electronics in order to have the ability to access every single waveguide (fibre) randomly, b) inability to perform label-free real-time signal detection.

These problems are solved by the present invention as described herein.

Summary of the invention

The innovations of the present invention in respect of the above prior art documents are achieved through the following features, which at the same time provide distinct advantages in terms of functionality and diagnostic potential:

a) A single node output for the readout of all optocouplers of the device. Such a configuration greatly facilitates the data collection process and greatly simplifies control electronics since a single readout electronics chain is needed and the multiplexing is enabled by selecting one emitter at a time.

b) Label-free detection is enabled in real time of analytes by exploiting the photon extinction shifts in spectral content and magnitude when biomolecules bind on noble nanoparticles immobilized on the waveguide surface. Alternatively, label-free detection can be achieved by introducing on the waveguide surface patterns of another dielectric that make the optocouplers coupling efficiency sensitive to the effective refractive index of the superstrate.

In a first aspect, the invention provides a monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light-emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light-emitting elements; and one single optical detector optically coupled to each of the plurality of optical waveguides, wherein the optical biosensor is adapted to detect an analyte by binding of the analyte on a surface of one or more of the waveguides and thereby changing the optical coupling between the optical detector and one or more of the light emitting elements.

In a second aspect, the invention provides a monolithic optical biosensor for sensing biomolecules, comprising: a light emitting element; an optical waveguide optically coupled to the light emitting element; and an optical detector optically coupled to the optical waveguide, the optical waveguide having a plurality of surface features, the surface features being adapted to effect a change in the optical coupling between the light emitting element and the optical detector when biomolecules to be sensed are provided over the waveguide.

In a third aspect, the invention provides a method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light-emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light-emitting elements; and one single optical detector optically coupled to the plurality of optical waveguides, wherein the optical biosensor device is adapted to detect an analyte by binding of the analyte on a surface of one or more of the waveguides and thereby changing the optical coupling between the optical detector and one or more of the light emitting elements; applying a solution containing the analyte to be sensed to the surface of one or more of the waveguides; and detecting a change of optical coupling between one or more of the light- emitting elements and the optical detector by measuring a change in detected light from one or more of the light-emitting elements by the optical detector.

In a fourth aspect, the invention provides a method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a light emitting element; an optical waveguide optically coupled to the light emitting element; and an optical detector optically coupled to the optical waveguide, the optical waveguide having a plurality of surface features, the surface features being adapted to effect a change in optical coupling between the light- emitting element and the optical detector when biomolecules to be sensed are provided over the waveguide; applying biomolecules to be sensed over the surface of the waveguide; and detecting a change of optical coupling between the light-emitting element and the optical detector by measuring a change in detected light from the light-emitting element by the optical detector.

In a fifth aspect, the invention provides a method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light -emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light emitting elements; and one single optical detector optically coupled to the plurality of optical waveguides, the optical waveguides having a plurality of surface features, the surface features being adapted to effect a change in optical coupling between the light- emitting elements and the single optical detector when biomolecules to be sensed are provided over the waveguides; applying biomolecules to be sensed over the surface of the waveguides; and detecting a change of optical coupling between the light-emitting elements and the optical detector by measuring a change in detected light one at a time from the light-emitting elements by the optical detector.

Detailed description of the invention

Disclosed herein is a multianalyte biosensor based on an array of monolithically integrated optoelectronic silicon transducers comprising avalanche diode light sources, silicon nitride optical fibers with multiplexed output signal on a single detector which is fabricated following silicon integrated circuit methods and can detect simultaneously multiple analytes in the same sample through either: the use of appropriately labeled biomolecules or surface nano-engineering that enables label-free detection of binding reactions in real time due to the change of the optical coupling between the integrated light sources and the integrated single node detector that is caused by the binding of the analytes on their respective recognition-molecules which have been previously immobilized on the plain (or nano-engineered) surface of the optical fibers that connect the light sources with the detector.

The present invention has certain novel features that differentiate it from the prior art and at the same time provide distinct advantages in terms of functionality and diagnostic potential:

Advantageously, the invention presents a single node output for the readout of all optocouplers.

Further, the invention may also enable label-free detection in real time of analytes by exploiting the photon extinction shifts in spectral content and magnitude when biomolecules bind on noble nanoparticles immobilized on the waveguide surface.

Alternatively, label-free detection can be achieved by introducing on the waveguide surface patterns of another dielectric that make the optocouplers' coupling efficiency sensitive to the effective refractive index of the superstrate.

An important element of one aspect of the present invention is surface nanoengineering of the fiber surface with isolated gold nanoparticles that permits label-free detection of bioreactions in real time.

Preferred embodiments of the invention are illustrated in the Figures, wherein:

Figure Ia shows a top view schematic of a set of monolithic optocouplers sharing the same detector node 13 ;

Figure Ib shows the cross section of the single p/n detector 13 where a number of independent waveguides 12 converge;

Figure 2 presents a schematic diagram of one of the optocouplers after modification of the waveguide 23 with the metal nanoparticles 24; Figure 3 shows a schematic diagram showing surface patterns 34 on the waveguide 23 that make the optocouplers coupling efficiency sensitive to the effective refractive index of the superstrate;

Figure 4 presents the signal changes obtained from an optical waveguide with surface modification as in Fig. 2, during adsorption of anti-mouse IgG antibody on to gold nanoparticles, immunoreaction with mouse IgG and irnmunoreaction of the immobilized mouse IgG with anti-mouse IgG antibody in solution; and

Figure 5 presents signal changes obtained from an optical waveguide with surface modification as in Fig. 3 during flow of distilled water followed by phosphate buffered saline reflecting changes in the effective refractive index of the waveguide superstrate medium.

The number of optocouplers shown in Fig. Ia is only indicative and can be expanded to any number with limits imposed only by the size of the chip, lithography and optocoupler layout. Such a configuration greatly facilitates the data collection process and greatly simplifies control electronics since a single readout electronics chain is needed and the multiplexing is enabled by selecting one emitter at a time. To enable this, the light emitting elements 11 are preferably adapted to be independently selected, so that the output from the detector 13 may be indicative of a selected one or more of the waveguides 12. The light emitting elements 11 of the present invention are preferably avalanche diode light sources, although other light sources may also be suitable.

For simplicity reasons, in Fig Ib only the waveguides 12 coming from left, right and from top are shown. The different waveguides deliver their photons independently to the detector p/n junction mainly at the abrupt breaking point of the waveguide at the edge of the p/n junction. By coupling light to each waveguide 12 at a time, a large number of optocouplers can be measured with a single detector 13.

Preferably, each waveguide 12 has a light emitting element 11 optically coupled at a first end and an optical detector 13 optically coupled at a second end. However, other arrangements may be possible, where light emitting elements and optical detectors are optically coupled at intermediate positions along the length of a waveguide.

In general, it is to be understood that light emitted by the light emitting elements of the present invention may be of a wavelength outside the normal visible spectrum, for example in the infra-red or ultraviolet regions.

In Figure 2 is presented a schematic of one of the optocouplers after modification of the waveguide 23 with noble metal nanoparticles 24. The nanoparticles 24 are functionalized with capture biomolecules that bind counterpart biomolecules and thereby change the nanoparticle photon extinction annihilation characteristics in spectral content and magnitude. This change is measured by monitoring the detector photocurrent changes with biomolecular binding. Binding events occurring between biomolecules close to the surface of a noble metal nanoparticle increase the refractive index of the nanoparticle' s immediate environment and cause a red shift of the homogeneous nanoparticle plasmon resonance [D. Eck, CA. Helm, Langmuir 17, 957-960,2001 ;A. Nichtl, K. Kulrzinger, Nanoletters, 3, 935-938, 2003) D. Eck, CA. Helm, Langmuir 17, 957-960,2001], especially when one binding molecule is attached to the surface of the gold nanoparticle. This causes a decrease of the detector photocurrent. In the present invention we propose for the first time a new methodology for the controlled fiber surface modification with gold nanoparticles based on the self- assembly of colloidal gold-streptavidin conjugate with immobilized biotin or biotinylated bovine serum albumin on the surface of the fiber. Although the present invention is described in relation to gold nanoparticles, it is to be understood that other noble metals may also be suitable, provided appropriate modifications are made to the process. Other noble metals may for example be selected from the group consisting of gold, silver, copper and the platinum group metals platinum, palladium, osmium, iridium, ruthenium and rhodium. According to the methodology, the transducers are first cleaned and hydrophylized in oxygen plasma. Then the transducers are immersed in solution of aminosilane or other silane that exposes reactive chemical groups for coupling of biomolecules or make the surface appropriate for adsorption of biomolecules. After that biotinylated bovine serum albumin or other biotinylated protein molecule is adsorbed onto the surface of the fiber. Then, the free binding sites on the surface of the fiber are covered with a solution of bovine serum albumin. After that, an appropriate microfluidic module adapted to transfer solutions over the fibers while insulating the electrical contact pads is applied on top of the transducer. Through this fluidic module a streptavidin colloidal gold conjugate is pumped and the output signal current of the transducer is monitored. When a 20-30% drop of the detector photocurrent is achieved the transucer is washed extensively with doubly distilled water and then oxidized in oxygen plasma. This way single plain gold colloidal nanoparticles are created on the fiber surface. The so prepared gold particles can couple proteins or thiolated DNA oligonucleotides through self assemply of the thiol groups on the gold surface whereas the oxidized silicon nitride or silicon oxide layer surface do not adsorb biomolecules for a certain period of time. Other deposition methods such as sputtering or adsorption of colloidal gold particles from solution could be used for surface nanoengineering of the fibers preferably provided that a decrease of the transducer photocurrent caused by the immobilized nanoparticles is in the range of 70-80% of its initial value (i.e. prior to nanoparticle application). Another important innovation of the present invention that will permit label-free detection is achieved by introducing on the waveguide surface patterns of another dielectric material that makes the optocouplers' coupling efficiency sensitive to the effective refractive index of the superstrate.

In Figure 3 a schematic drawing shows surface patterns 34 on the waveguide 23 that make the optocouplers coupling efficiency sensitive to the effective refractive index of the superstrate. Fabrication of the grating involves a chemical vapor deposition step of SiO₂ over the Silicon Nitride waveguide followed by lithography and etching of the expose regions. Since these periods are quite long, the etching can be performed through wet chemistry, which has the additional advantage of good selectivity between the silicon dioxide (high etching rate) and the silicon nitride (much lower etching rate). In the device shown in Figure 3, the waveguided modes enter from regions with no cladding layer 33 to regions with a cladding layer 33. Each time the photons cross a different region reflection losses are experienced so that the photocurrent at the detector 22 is reduced with respect to the case of a uniform cladding layer. The refractive index of the superstrate affects the reflection coefficient and the overall transmission constant. Therefore, measuring the detector photocurrent changes with different superstrate media or overlayers provides a measure for the effective refractive index of the overlaying medium. Therefore if biomolecules bind on the exposed waveguide 34 either directly or indirectly through another immobilized biomolecule, the detector 22 should sense a different photocurrent since the effective refractive index in the vicinity of the exposed waveguide surface should increase. In fact it should sense an increase because the effective refractive index of the exposed regions (no SiO₂ cladding) will move closer to the refractive index of the SiO₂ cladding regions (1.46). It is desirable to have make the length of such regions (with cladding and with no cladding) relatively long (>10 μn) to make the fabrication process even more relaxed in terms of lithography requirements and also to exploit the advantages of such long period gratings (LPGs) (X. Daxhelet, M. Kulishov, Optics Letters 28, 2003; S.W.James & R.P. Tatam, Meas. Sci. Technol. 14 R49- R61, 2003; I. Ishaqa et al. Sensors and Actuators B 107, 738-741, 2005). As in LPGs, here we also couple light from the propagating core mode to the surrounding U medium modes which are lossy and depend on the effective medium refractive index.

An additional reason for the long period grating is to contain the overall transmission coefficient into a realistic range so that enough light arrives at the detector.

In a general aspect, a waveguide of the invention is adapted to detect biomolecules provided over the waveguide. Preferably, biomolecules are provided by passing an analyte solution over the waveguide such that the biomolecules therein are able to bind or otherwise associate with the waveguide surface or with surface features provided thereon and to consequently influence the optical path between a light emitting element and a detector coupled to the waveguide. The optical path may be influenced by refraction, diffraction, absorption or other changes in light interactions resulting from the presence of biomolecules proximate the waveguide which thereby affect the level of light received by the optical detector.

The invention is further illustrated through the following examples:

Example 1

On a wafer with a device in the form as illustrated in figure 1, a microfiuidic device is applied that has been appropriately designed in order to allow supply of reagents solution in all fibers of a single device simultaneously while it insulates the contact pads. Through this microfiuidic module all the reagents solutions were run and the detector output signal recorded with the sampling rate of 1 sample/second (Figure 2). First, a 50 mM phosphate buffered saline, pH 7.4 (PBS buffer) was pumped onto the surface in order to establish a baseline for 3 min at a rate of 20 d/min (arrow 1). Then a 66.67 nM anti-mouse IgG antibody solution in PBS buffer was injected (arrow 2) and run with a constant rate of 20 d/min for 30 min at room temperature (RT). When the reaction reached a plateau, the device was washed with PBS for 3 min (arrow 3) followed by injection of a 10 nM mouse IgG solution in PBS (arrow 4) that was run for 30 min at a rate of 20 d/min. After completion of this reaction, washing with PBS buffer for 3 min followed (arrow 5) and a 30 nM anti-mouse IgG antibody solution in PBS buffer was introduced (arrow 6) and run for 30 min at RT at the same rate. Finally, the device was washed with PBS buffer (arrow 7). As it shown in Figure 3, all the steps, including immobilization of anti-mouse IgG antibody onto the device surface (arrows 2-3), immunoreaction of the mouse IgG with the immobilized antibody (arrows 4-5) and binding of the anti-mouse IgG antibody onto mouse IgG (arrows 6-7), are clearly distinguished on the recording of the detector output. The immobilization of the anti-mouse IgG onto the fiber was completed in approximately 15 min. The reaction of mouse IgG with the immobilized antibody is a slower reaction that requires more than 30 min to reach a plateau, whereas the reaction of the immunoadsorbed mouse IgG with the liquid phase anti-mouse IgG is completed in approximately 25 min.

Example 2

On a wafer with a device in the form shown in Figure 1, modified according to Figure 3, a microfluidic device described in Example 1 is applied. Through this fluidic device distilled water is pumped for 80 sec at a rate of 20 μVmin (Figure 5, arrow 51) over the waveguides of the array of the transducers and the output signal of the detector is recorded with a sampling rate of 1 sample/second. Then, a 50 mM phosphate buffered saline, pH 7.4, was pumped over the waveguides (arrow 52). A change of the output signal is clearly demonstrated, indicating that the proposed surface modification of the waveguides is sensitive to refractive index changes.

Claims

1. A monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light-emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light-emitting elements; and one single optical detector optically coupled to each of the plurality of optical waveguides, wherein the optical biosensor is adapted to detect an analyte by binding of the analyte on a surface of one or more of the waveguides and thereby changing the optical coupling between the optical detector and one or more of the light emitting elements.

2. The monolithic optical biosensor of claim 1 wherein each of the plurality of light emitting elements is adapted to be independently selected.

3. The monolithic optical biosensor of claim 1 wherein the biosensor is configured to simultaneously detect multiple analytes.

4. The monolithic optical biosensor of any of claims 1 to 3 wherein the optical biosensor is configured to detect an analyte bound to a surface of the one or more waveguides by interaction with labelled biomolecules.

5. The monolithic optical biosensor of any of claims 1 to 4 wherein one or more of the optical waveguides comprise a plurality of surface features adapted to effect a change in an effective refractive index of a portion of the waveguide proximate thereto when a solution of biomolecules to be sensed is provided over the waveguide.

6. The monolithic optical biosensor of claim 5 wherein the surface features comprise nanoparticles.

7. The monolithic optical biosensor of claim 6 wherein the nanoparticles comprise capture biomolecules adapted to bind counterpart biomolecules.

8. The monolithic optical biosensor of claim 6 or claim 7 wherein the nanoparticles are formed of a noble metal.

9. The monolithic optical biosensor of claim 8 wherein the noble metal is gold.

10. The monolithic optical biosensor of claim 5 wherein the surface features comprise a surface pattern of a material having a refractive index differing from a refractive index of a material comprising the one or more waveguides.

11. The monolithic optical biosensor of claim 10 wherein the surface pattern is a periodic photonic formation.

12. The monolithic optical biosensor of claim 10 wherein the material forming the surface pattern comprises silicon dioxide and the material forming the waveguide comprises silicon nitride.

13. A monolithic optical biosensor for sensing biomolecules, comprising: a light emitting element; an optical waveguide optically coupled to the light emitting element; and an optical detector optically coupled to the optical waveguide, the optical waveguide having a plurality of surface features, the surface features being adapted to effect a change in the optical coupling between the light emitting element and the optical detector when biomolecules to be sensed are provided over the waveguide.

14. The monolithic optical biosensor of claim 13 wherein the change in optical coupling is effected by a change in an effective refractive index of a portion of the waveguide proximate the surface features.

15. The monolithic optical biosensor of claim 13 wherein the surface features comprise nanoparticles.

16. The monolithic optical biosensor of claim 15 wherein the nanoparticles comprise capture biomolecules adapted to bind counterpart biomolecules.

17. The monolithic optical biosensor of claim 15 or claim 16 wherein the nanoparticles are formed of a noble metal.

18. The monolithic optical biosensor of claim 17 wherein the noble metal is gold.

19. The monolithic optical biosensor of claim 13 wherein the surface features comprise a periodic photonic formation of a material having a refractive index differing from a refractive index of a material comprising the one or more waveguides.

20. The monolithic optical biosensor of claim 19 wherein the surface features comprise a periodic photonic formation.

21. The monolithic optical biosensor of claim 19 wherein the material forming the surface pattern comprises silicon dioxide and the material forming the waveguide comprises silicon nitride.

22. A method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light-emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light-emitting elements; and one single optical detector optically coupled to the plurality of optical waveguides, wherein the optical biosensor device is adapted to detect an analyte by binding of the analyte on a surface of one or more of the waveguides and thereby changing the optical coupling between the optical detector and one or more of the light emitting elements; applying a solution containing the analyte to be sensed to the surface of one or more of the waveguides; and detecting a change of optical coupling between one or more of the light- emitting elements and the optical detector by measuring a change in detected light from one or more of the light-emitting elements by the optical detector.

23. A method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a light emitting element; an optical waveguide optically coupled to the light emitting element; and an optical detector optically coupled to the optical waveguide, the optical waveguide having a plurality of surface features, the surface features being adapted to effect a change in optical coupling between the light- emitting element and the optical detector when biomolecules to be sensed are provided over the waveguide; applying biomolecules to be sensed over the surface of the waveguide; and detecting a change of optical coupling between the light-emitting element and the optical detector by measuring a change in detected light from the light-emitting element by the optical detector.

24. A method of sensing biomolecules, comprising: providing a monolithic optical biosensor for sensing biomolecules, comprising: a plurality of light -emitting elements; a plurality of optical waveguides, each waveguide optically coupled to a respective one of the plurality of light emitting elements; and one single optical detector optically coupled to the plurality of optical waveguides, the optical waveguides having a plurality of surface features, the surface features being adapted to effect a change in optical coupling between the light- emitting elements and the single optical detector when biomolecules to be sensed are provided over the waveguides; applying biomolecules to be sensed over the surface of the waveguides; and detecting a change of optical coupling between the light-emitting elements and the optical detector by measuring a change in detected light one at a time from the light- emitting elements by the optical detector.