Immunoassay devices and methods of using same

An immunoassay cartridge for sensing at least one analyte in a biological sample is disclosed. The immunoassay cartridge comprises a supporting plate, a reaction cavity made within the supporting plate and having at least one analyte-binding molecule immobilized therein, a sample receiving end connected to the reaction cavity to allow the biological sample to flow into the reaction cavity for forming at least one complex of the analyte and the analyte-binding molecule, a first package containing a recognizing molecule, a first channel communicating the first package and the reaction cavity to allow the recognizing molecule to flow into the reaction cavity for forming a first product of the complex and the complex-binding molecule, a second package containing a buffer solution and a second channel communicating the second package and the reaction cavity to allow the buffer solution to flow into the reaction cavity.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to immunoassay devices and methods of using same. More particularly, the present invention relates to immunoassay devices capable of being accommodated by an analyzer for automatic immunoassays.

BACKGROUND OF THE INVENTION

Immunoassays using immobilized immunoreagents are in widespread use for the detection and quantification of biological molecules in samples. An immunoreagent, which may comprise an antibody or antigen, is first immobilized on a solid surface, such as a test tube, microplate, beads or the like. The immobilized reagent is then contacted with a sample solution containing a complementary immunoreagent, whereby an immobilized immunocomplex is formed between the immunoreagent and complementary immunoreagent. The immobilized immunocomplex can be separated from the unreacted sample solution, preferably with repeated washing of the immobilized immunocomplex. The separated immunocomplex may be subjected to further processing to quantitate the amount of the immunocomplex. Quantitative methods include, for example, radioimmunoassay (RIA), wherein the amount of the adsorbed immunocomplex may be determined by counting radioactive disintegrations; enzyme-linked immunosorbent assay (ELISA), wherein the adsorbed immunocomplex, which has an enzyme coupled thereto, is contacted with a substrate for the enzyme to produce a detectable product; immunofluorescent assay, wherein the fluorescent intensity of a fluorescent substance linked to the immunocomplex is measured; and chemiluminescent assay wherein the chemiluminescence of a chemiluminescent agent is measured.

Processes of the immunoassays, however, take time and resources. In addition, errors generated during the complex immunoassay accumulate gradually. For example, the enzymatic immunoassay is conducted by measuring the fluorescence intensity of the substrate after a certain period of time, either stopping the enzyme reaction with a stopping solution or not. In these steps, the duration from the initiation of enzyme reaction to the measurement should be strictly controlled, and so-called zero point correction is needed for the fluorescence intensity of the substrate because the intensity at the starting point is not always zero.

Except above descriptions, a researcher or an operator usually needs to load samples, pipette reagents or add a substrate into a test tube upon a bench, pour the liquid inside the test tube directly over the lab sink, and place the test tube into a machine, e.g. the ELISA reader, when the immunoassay has been initiated. Accordingly, it takes time and energy for the researcher or the operator to complete the assay. In addition, contaminations cannot effectively avoided as long as the assay is carried out by humans.

SUMMARY OF THE INVENTION

The present invention discloses immunoassay cartridges for automatically detecting at least one analyte in a biological sample. One of the immunoassay cartridges comprises a supporting plate, a reaction cavity made within the supporting plate and having at least one analyte-binding molecule immobilized therein, a sample receiving end connected to the reaction cavity to allow the biological sample to flow into the reaction cavity for forming at least one complex of the analyte and the analyte-binding molecule, a first package containing a recognizing molecule, a first channel communicating the first package and the reaction cavity to allow the recognizing molecule to flow into the reaction cavity for forming a first product of the complex and the complex-binding molecule, a second package containing a buffer solution and a second channel communicating the second package and the reaction cavity to allow the buffer solution to flow into the reaction cavity.

Another immunoassay cartridge comprises a tank for containing a buffer solution, a supporting plate covered onto the tank to form an enclosure, a reaction cavity made within the cover plate and having at least one analyte-binding molecule immobilized therein, a conduit communicating the tank and the reaction cavity to allow the buffer solution to spill into the reaction cavity, a sample loading chamber for receiving the biological sample, a first channel communicating the sample loading chamber and the reaction cavity to allow the biological sample to flow into the reaction cavity for forming at least one complex of the analyte and the analyte-binding molecule; a first package containing a recognizing molecule and a second channel communicating the first package and the reaction cavity to allow the recognizing molecule to flow into the reaction cavity for forming a first product of the complex and the recognizing molecule.

The present invention discloses an immunoassay cartridge for sensing at least one analyte in a biological sample. The cartridge comprises a tank for containing a buffer solution, a supporting plate covered onto the tank to form an enclosure, a reaction cavity made within the cover plate and having at least one analyte-binding molecule immobilized therein, a conduit communicating the tank and the reaction cavity to allow the buffer solution to spill into the reaction cavity, a sample loading chamber for receiving the biological sample, a first channel communicating the sample loading chamber an the reaction cavity to allow the biological sample to flow into the reaction cavity for forming at least one complex of the analyte and the analyte-binding molecule, a first package within the supporting plate containing a recognizing molecule and a second channel communicating the first package and the reaction cavity to allow the recognizing molecule to flow into the reaction cavity for forming a first product of the complex and the recognizing molecule.

EXAMPLES

Referring toFIG. 1, a side view of an immunoassay cartridge100is shown herein. The immunoassay cartridge100includes a plate structure110and an extending portion120coupled thereto. In an embodiment of the present invention, the extending portion120and the plate structure110are made by a procedure of one-piece molding. In another embodiment, the extending portion120and the plate structure110are fabricated separately and then assembled together.

Within the plate structure110includes a reaction cavity130, and at least one analyte-binding molecule is immobilized on the bottom surface of the reaction cavity130. In one preferred embodiment of the present invention, the analyte-binding molecule(s) within the reaction cavity130is/are printed to from an array. In addition, the method of immobilizing the analyte-binding molecule in the reaction cavity130comprises physical adsorption, cross-linking, covalent binding, entrapment or any combination thereof.

Methods of immobilization are described in detail below. However, it is appreciated that they are not used to limit the score and spirit of the present invention.

In one of these methods, a plateform made of poly (methylmethacrylate) (PMMA) is used as the bottom surface of the reaction cavity130and firstly derivatized by following a procedure similar to that described by Karandikar et al. at polym prepr 30:250-251 at 1989 with minor modifications. PMMA is well suited for use as a plateform for microfabricated devices because of its high dielectric constant, transparency, thermal conductivity that is comparable to silica, low cost, and ease of microfabrication.

Next, butyllithium (1 mmol) was transferred by cannula into a nitrogen-purged vessel containing 6 mmol of dry ethylenediamine or 1,3-diaminopropane. The resulting product of the addition of the ethylenediamine to butyllithium was dark purple, and upon addition of the 1,3-diaminopropane to butyllithium the product was yellowish-brown. After the reaction was stirred for 3 h, the lithiated diamine was added to a nitrogen-purged vessel containing the PMMA plateform to be derivatized. The reaction was quenched after 8 min with copious amounts of deionized water and the PMMA plateform were ready to be funtionalized.

The procedure for functionalization was similar to that of Locascio-Brown et al. published at Anal Chim Acta 228:107-116 at 1990 with small adjustments. An aqueous solution of 5% (v/v) glutaric dialdehyde was added to the derivatized PMMA plateform and shaken for 4 h. Afterward the supernatant was discarded and the PMMA plateform were washed with deionized water several times. To immobilize the analyte-binding molecule, a reaction mixture containing the analyte-binding molecule, 50 mM Tris-HCl, 10 mM MgCl2, 20% glycerol, and 50 mM sodium chloride was printed onto the bottom surface of the reaction cavity130and incubated for 3 h at 37° C. The reaction cavity130was then washed with 50 mM monobasic sodium phosphate buffer (may contain surfactant such as 0.5% Tween 20), deionized water, and stored in an appropriated buffer at 4° C. until use.

Another method of immobilizing the analyte-binding molecule on a silicate-based plateform is described. Firstly, the cleaned plateform was washed with sodium-dried toluene and then immersed in a solution of 10% 3-aminopropyltriethoxysilane (APTES) in dried toluene overnight at room temperature (RT). After removal of the solution, the plateform was rinsed several times with toluene and acetone and dried in an oven at 110° C. for 1 h. The dried plateform was immersed in 100 mL ethanol and sonicated for 10 min to introduce the solvent into the pores.

After washing with borate buffer (BB), the amine groups of the APTES silanized plateform was reacted with 2.5% v/v glutaraldehyde (GA) in BB for 1 h at RT, followed by thorough rinsing with deionized water in order to remove traces of glutaraldehyde to avoid cross-linking after addition of the analyte-binding molecule. Analyte-binding molecule (1 mg/mL) in BB was added to the GA-activated plateforms and reacted overnight at 4° C. under gentle shaking. After 12 h, the residual aldehyde groups remaining after analyte-binding molecule attachment were blocked with 10 mg/mL of L-lysine. The Schiff bases were reduced with 20 mg/mL NaBH3CN solution in BB, and the plateforms were allowed to proceed for 1-2 h under stirring at RT. The plateforms were then carefully washed and stored in 0.1 M Tris/HCl buffer at 4° C. until use.

For the silicate-based plateform, another method of immobilization is described herein. The cleaned microchips were immersed in 0.5% v/v solution of BPEI (branched polyethylenimine) in BB and kept under stirring at RT overnight and then thoroughly washed with BB. To incorporate active aldehyde groups, the plateform was reacted with 2.5% v/v GA in BB for 2 h at RT under stirring. After careful washing with deionized water and BB, the aldehyde-functionalized plateforms were printed with analyte-binding molecule solution at the concentration of 0.5 to 1 mg/mL for overnight at 4° C. Then residual aldehyde groups on the plateform were blocked and the Schiff bases reduced as described above.

Another method of immobilizing the analyte-binding molecule is described herein. The cleaned plateform was reacted with 3-glycidoxypropyltrimethoxysilane (GOPS) in dry toluene, containing 2% v/v GOPS and 0.2% triethylamine at RT. After 1 h, the GOPS-coated plateform was first rinsed with toluene, then with acetone, and then dried in an oven at 110° C. for 1 h. To introduce a solvent in the pores, the plateform was sonicated in 100 mL of ethanol for 10 min and then were washed with deionized water. A solution of 0.5% v/v BPEI in succinate buffer was added, and the reaction mixture was gently shaken for 5 h at RT. After careful washing with deionized water, the plateform was treated with 2.5% v/v GA in BB. After 2 h, the microchips were rinsed and then immersed in analyte-binding molecule solution in BB with the concentration of 0.5 to 1 mg/mL. The reaction was allowed to proceed overnight at 4° C., after which the plateform was blocked and reduced as described above.

Still another method of immobilization is described herein. Two approaches were used to immobilize the analyte-binding molecule on to the surfaces of the PDMS plateform: namely, (1) passive adsorption on the surface; and (2) site-selective binding to immobilized analyte-binding molecule. In the first approach, 10 mg/ml of analyte-binding molecules were passively adsorbed on to the surfaces of the PDMS plateform from a 0.1 M PBS buffer solution (pH 7.4) for 90 min. This was followed by a blocking step for 1 h. The composition of the blocking buffer used was 0.5% BSA (w/v), 0.5% casein (w/v) and 0.5% Tween 20 (v/v) in PBS. In the second approach, PDMS plateform was functionalized with analyte-binding molecule in the following three-step process: first, the surface was coated with BSA (1 mg/ml in sodium phosphate buffer) for 3.5 h. Second, the BSA-coated surface was then activated with 0.1% GA in pure water for 1 h. Finally, analyte-binding molecule (20 mg/ml in sodium phosphate buffer) was covalently bound via the GA groups. No blocking step was used following the attachment of the capture antibody molecules. All experiments were carried out at room temperature.

One end161of a sample receiving end160is connected to the reaction cavity130. The other end162of the sample receiving end160penetrates through the extending portion120and has an opening163on a sidewall1201of the extending portion120. In this embodiment, the extending portion120is provided to protect the sample receiving end160from being broken.

A biological sample suspected of an analyte is received from the opening163and flows into the reaction cavity130via the sample receiving end160. In another embodiment, the biological sample is loaded into the reaction cavity130. That is, the sample receiving end160can be optionally omitted from the immunoassay cartridge100according to practical needs. The biological sample preferably comprises a blood sample, a serum sample or a saliva sample. In addition, the sample receiving end160is a capillary.

In one preferred embodiment of the present invention, the analyte is an antigen and the analyte-binding molecule is a first antibody correspondingly. In another embodiment, the analyte-binding molecule is a ligand when the analyte is a protein.

The plate structure110comprises a first package140and a second package150filled with a recognizing molecule and a buffer solution, respectively. It is noted that the recognizing molecule is preferably a second antibody when the analyte is the antigen and the analyte-binding molecule is the first antibody. However, it is appreciated that these are not used to limit the score and the spirit of the present invention.

As shown in the figure, both of the first package140and the second package150are adjacent to the reaction cavity130. A first channel170is used to communicate the first package140and reaction cavity130, whereas a second channel180is used to communicate the second package150and reaction cavity130.

In one preferred embodiment of the present invention, the first package140and the second package150comprise pits fabricated within the plate structure110and coverings covered onto the pits. In another embodiment, the first package140and the second package150comprise containers coupled to the plate structure110and coverings covered onto the containers.

The first channel170and the second channel180are both sealed by stoppers. When the covering of the first package140or the second package150is pressurized, the recognizing molecule or the buffer solution inside the respective first package140or the second package150breaks through the stopper and flows into the reaction cavity130. In the embodiment, the covering is made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper could be made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

The immunoassay cartridge100further comprises a removable lid190capable of being covered onto the plate structure110. On the inner surface of the removable lid190is lined with an adsorption pad195. In one preferred embodiment of the present invention, the adsorption pad195is made of adsorbing materials like membranes, tissues, or polymers such as starch-acrylic acid graft copolymer, copoly (vinyl alchol-acrylic acid), cross-linking copolyacrylic acid or combinations thereof. It is appreciated that these are not used to limit the score and the spirit of the present invention.

When the immunoassay cartridge100is accommodated by an analyzer (not shown herein), the biological sample flowing into the reaction cavity130through the sample receiving end160reacts with the analyte-binding molecule immobilized in the reaction cavity130, thus forming a complex of the analyte and the analyte-binding molecule.

Thereafter, the removable lid190is covered onto the plate structure110to allow the adsorption pad195attached thereinside to adsorb the liquid in the reaction cavity130. Next, the removable lid190is lifted to leave the plate structure110.

It is noted that the coverings of the first package140and the second package150should be avoided swelling out of the upper surface1101of the plate structure110when the first package140and the second package150are loaded with the recognizing molecule and the buffer solution. Accordingly, the recognizing molecule and the buffer solution inside the first package140and the second package150will not be squeezed into the reaction cavity130while covering the removable lid190onto the plate structure110.

After forming the complex of the analyte and the analyte-binding molecule, the buffer solution in the second package150is automatically pressed to break the stopper and flow into the reaction cavity130via the second channel180for a purpose of wash.

Next, the used adsorption pad195should be replaced with a fresh one by the analyzer for avoiding contaminations. That is, the used adsorption pad195should be replaced with a fresh one every time when it is used to adsorb the liquid within the reaction cavity130.

After that, the removable lid190is covered to the plate structure110to allow the new adsorption pad195attached thereinside to adsorb the liquid in the reaction cavity130.

In another embodiment, the adsorption pad195is directly installed in the analyzer instead of being attached on the removable lid190. The purpose of the adsorption pad195is to adsorb the liquid within the reaction cavity130rapidly. Accordingly, various possible modifications and substitutions, without departing from the purpose, should be included in the present invention. After evacuating the liquid within the reaction cavity130using the adsorption pad195, the removable lid190is removed from the plate structure110.

Next, the recognizing molecule such as a gold-conjugated antibody in the first package140is automatically forced to break the stopper and flow into the reaction cavity130via the first channel170, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid190is covered to the plate structure110to allow the adsorption pad195attached thereinside to adsorb the liquid in the reaction cavity130. The removable lid190is then removed from the plate structure110. Lastly, the second package150is automatically pressed again to force the buffer solution to flow into the reaction cavity130for rinse.

It is noted that, in another embodiment, diameters of the first channel170and the second channel180are designed to allow the recognizing molecule and the buffer solution to stay in the first package140and the second package150and then flow into the reaction cavity130when the first package140and the second package150suffer the pressure. That is, the stopper described above is absent in this embodiment.

The plate structure110in this example optionally comprises a third package197filled with a signal molecule. In one preferred embodiment of the present invention, the third package197comprises a pit made within the plate structure110and a covering covered onto the pit. In another embodiment, the third package197comprises a container coupled to the plate structure110and a covering covered onto the container.

In the enzymatic immunoassay, the complex of the analyte and the analyte-binding molecule is bound with an enzyme. The enzyme-bound complex is then contacted with the signal molecule, e.g. a substrate, for the enzyme to produce a detectable substance.

As described above, a third channel198communicates the third package197and the reaction cavity130. Accordingly, the third package197is adjacent to the reaction cavity130.

In one preferred embodiment of the present invention, the third package197comprises a pit fabricated within the plate structure110and a covering covered onto the pit. Further, the third channel198is sealed by a stopper. When the covering of the third package197is pressurized, the signal molecule inside the third package197breaks through the stopper and flows into the reaction cavity130. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

After forming the first product of the complex and the recognizing molecule and washing the reaction cavity130with the buffer solution, the signal molecule in the third package197is automatically pressed to break the stopper and flow into the reaction cavity130via the third channel198, thus forming a second product of the first product and the signal molecule. Subsequently, the removable lid190is covered to the plate structure110to allow the adsorption pad195attached thereinside to adsorb the liquid in the reaction cavity130. Then, the removable lid190is removed from the plate structure110.

Lastly, the second package150is automatically pressed again to force the buffer solution to flow into the reaction cavity130for rinse. The second product is subjected to detect the concentration of the analyte in the biological sample.

It is noted that, in another embodiment, a diameter of the third channel198is designed to allow the signal molecule to stay in the third package197and then flow into the reaction cavity130when the third package197is pressed. In other words, the stopper described above is absent herein.

Referring toFIG. 2, a side view of an immunoassay cartridge200is shown herein. The immunoassay cartridge200includes a plate structure210and an extending portion220coupled thereto. In an embodiment of the present invention, the extending portion220and the plate structure210are made by a procedure of one-piece molding. In another embodiment, the extending portion220and the plate structure210are fabricated separately and then assembled together.

Within the plate structure210includes a reaction cavity230, and at least one analyte-binding molecule is immobilized on the bottom surface of the reaction cavity230. In a preferred embodiment of the present invention, the analyte-binding molecule(s) within the reaction cavity230is/are printed as an array. In addition, the method of immobilizing the analyte-binding molecule in the reaction cavity230comprises physical adsorption, cross-linking, covalent binding, entrapment or any combination thereof.

One end261of a sample receiving end260is connected to the reaction cavity230. The other end262of the sample receiving end260penetrates through the extending portion220and has an opening263on a sidewall2201of the extending portion220. In this embodiment, the extending portion220is provided to protect the sample receiving end260from being broken.

A biological sample suspected of an analyte is received from the opening263and flows into the reaction cavity230via the sample receiving end260. In another embodiment, the biological sample is loaded into the reaction cavity230. That is, the sample receiving end260can be optionally omitted from the immunoassay cartridge200according to practical needs.

The biological sample preferably comprises a blood sample, a serum sample or a saliva sample. In addition, the sample receiving end260is a capillary.

In one preferred embodiment of the present invention, the analyte is an antigen and the analyte-binding molecule is a first antibody correspondingly. In another embodiment, the analyte-binding molecule is a ligand when the analyte is a protein.

The plate structure210comprises a first package240and a second package250filled with a recognizing molecule and a buffer solution, respectively. It is noted that the recognizing molecule is preferably a second antibody when the analyte is the antigen and the analyte-binding molecule is the antigen. However, it is appreciated that these are not used to limit the score and the spirit of the present invention.

Differences between Example 1 and Example 2 are that the immunoassay cartridge200in this example further comprises a first pipe270and a second pipe280. The first pipe270is used to communicate the first package240and reaction cavity230, whereas the second pipe280is served to connect the second package250and reaction cavity230. As shown in the figure, the first pipe270and the second pipe280are embedded within the plate structure210. It is appreciated that, however, this is not used to limit the score and the spirit of the present invention.

In one preferred embodiment of the present invention, the first package240and the second package250comprise pits fabricated within the plate structure210and coverings covered onto the pits. In another embodiment, the first package240and the second package250comprise containers coupled to the plate structure210and coverings covered onto the containers.

The first pipe270and the second pipe280are both sealed by stoppers. When the covering of the first package240or the second package250is pressurized, the recognizing molecule or the buffer solution inside the respective first package240or the second package250breaks through the stopper and flows into the reaction cavity230. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

As described in Example 1, the immunoassay cartridge200in this example also comprises a removable lid290capable of being covered onto the plate structure210. On the inner surface of the removable lid290is lined with an adsorption pad295. In one preferred embodiment of the present invention, the adsorption pad295is made of adsorbing materials like membranes, tissues, or polymers such as starch-acrylic acid graft copolymer, copoly (vinyl alchol-acrylic acid), cross-linking copolyacrylic acid or combinations thereof. It is appreciated that these are not used to limit the score and the spirit of the present invention.

When the immunoassay cartridge200is accommodated by an analyzer (not shown herein), the biological sample flowing into the reaction cavity230through the sample receiving end260reacts with the analyte-binding molecule immobilized in the reaction cavity230, thus forming a complex of the analyte and the analyte-binding molecule.

Thereafter, the removable lid290is covered onto the plate structure210to allow the adsorption pad295attached thereinside to adsorb the liquid in the reaction cavity230. Next, the removable lid290is removed from the plate structure210.

It is noted that the coverings of the first package240and the second package250should be avoided swelling out of the upper surface2101of the plate structure210when the first package240and the second package250are loaded with the recognizing molecule and the buffer solution. Accordingly, the recognizing molecule and the buffer solution inside the first package240and the second package250will not be squeezed into the reaction cavity230while covering the removable lid290onto the plate structure210.

After forming the complex of the analyte and the analyte-binding molecule, the buffer solution in the second package250is automatically pressed to break the stopper and flow into the reaction cavity230via the second pipe280for wash. Next, the used adsorption pad295should be replaced with a fresh one by the analyzer for avoiding contaminations. That is, the used adsorption pad295should be replaced with a fresh one every time when it is used to adsorb the liquid within the reaction cavity230.

After that, the removable lid290is covered to the plate structure210to allow the new adsorption pad295attached thereinside to adsorb the liquid in the reaction cavity230.

In another embodiment, the adsorption pad295is directly installed in the analyzer instead of being attached on the removable lid290. The purpose of the adsorption pad295is to adsorb the liquid within the reaction cavity230rapidly. Accordingly, various possible modifications and substitutions, without departing from the purpose, should be should be included in the present invention. After evacuating the liquid within the reaction cavity230using the adsorption pad295, the removable lid290is removed from the plate structure210.

Next, the recognizing molecule such as a gold-conjugated antibody in the first package240is automatically squeezed to break the stopper and flow into the reaction cavity230via the first pipe270, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid290is covered onto the plate structure210to allow the adsorption pad295attached thereinside to adsorb the liquid contained in the reaction cavity230. The removable lid290is then removed from the plate structure210. Lastly, the second package250is automatically pressed again to force the buffer solution to flow into the reaction cavity230for rinse.

It is noted that, in another embodiment, diameters of the first channel270and the second channel280are designed to allow the recognizing molecule and the buffer solution to stay in the first package240and the second package250and then flow into the reaction cavity230when the first package240and the second package250suffer the pressure. That is, the stopper described above is absent in this embodiment.

In one preferred embodiment of the present invention, the third package297comprises a pit made within the plate structure210and a covering covered onto the pit. In another embodiment, the third package297comprises a container coupled to the plate structure210and a covering covered onto the container.

In the enzymatic immunoassay, the complex of the analyte and the analyte-binding molecule is bound with an enzyme. The enzyme-bound complex is then contacted with the signal molecule, e.g. a substrate, for the enzyme to produce a detectable substance.

As described above, a third pipe298communicates the third package297and the reaction cavity230. Accordingly, the third package297is adjacent to the reaction cavity230.

In one preferred embodiment of the present invention, the third package297comprises a pit fabricated within the plate structure210and a covering covered onto the pit. Further, the third channel298is sealed by a stopper. When the covering of the third package297is pressurized, the signal molecule inside the third package297breaks through the stopper and flows into the reaction cavity230. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers. In another embodiment, the third package297is coupled to the plate structure210.

After forming the first product of the complex and the recognizing molecule and washing the reaction cavity230with the buffer solution, the signal molecule in the third package297is automatically pressed to break the stopper and flow into the reaction cavity230via the third channel298, thus forming a second product of the first product and the signal molecule. Subsequently, the removable lid290is covered to the plate structure210to allow the adsorption pad295attached thereinside to adsorb the liquid in the reaction cavity230. Then, the removable lid290is removed from the plate structure210.

Lastly, the second package250is automatically pressed again to force the buffer solution to flow into the reaction cavity230for rinse. The second product is subjected to detect the concentration of the analyte in the biological sample.

It is noted that, in another embodiment, a diameter of the third channel298is designed to allow the signal molecule to stay in the third package297and then flow into the reaction cavity230when the third package297is pressed. In other words, the stopper described above is absent herein.

Referring toFIG. 3, a side view of an immunoassay cartridge300is shown herein. The immunoassay cartridge300includes a plate structure310and an extending portion320coupled thereto. In an embodiment of the present invention, the extending portion320and the plate structure310are made by a procedure of one-piece molding. In another embodiment, the extending portion320and the plate structure310are fabricated separately and then assembled together.

Within the plate structure310includes a reaction cavity330, and at least one analyte-binding molecule is immobilized on the bottom surface of the reaction cavity330. In one preferred embodiment of the present invention, the analyte-binding molecule(s) within the reaction cavity330is/are printed to form an array. In addition, the method of immobilizing the analyte-binding molecule in the reaction cavity330comprises physical adsorption, cross-linking, covalent binding, entrapment or any combination thereof.

One end361of a sample receiving end360is connected to the reaction cavity330. The other end362of the sample receiving end360penetrates through the extending portion320and has an opening363on a sidewall3201of the extending portion320. In this embodiment, the extending portion320is provided to protect the sample receiving end360from being broken.

A biological sample suspected of an analyte is received from the opening363and flows into the reaction cavity330via the sample receiving end360. In another embodiment, the biological sample is loaded into the reaction cavity330. That is, the sample receiving end360can be optionally omitted from the immunoassay cartridge300according to practical needs.

The biological sample preferably comprises a blood sample, a serum sample or a saliva sample. In addition, the sample receiving end360is a capillary.

In one preferred embodiment of the present invention, the analyte is an antigen and the analyte-binding molecule is a first antibody correspondingly. In another embodiment, the analyte-binding molecule is a ligand when the analyte is a protein.

The immunoassay cartridge300further comprises a first package340within the plate structure310and a second package350under the plate structure310. The first package340is filled with a recognizing molecule, and the second package350is full of a buffer solution. In another embodiment, the first package340comprises a container coupled to the plate structure310and a covering covered onto the container.

The recognizing molecule is preferably a second antibody when the analyte is the antigen and the analyte-binding molecule is the first antibody. However, it is appreciated that these are not used to limit the score and the spirit of the present invention. One of differences between Example 3 and Example 1 is that the second package350in this example is located under the plate structure310.

A first channel370within the plate structure310communicates the first package340and reaction cavity330, whereas a second channel380connects the second package350and reaction cavity330. A diameter of the second channel380is designed to avoid the liquid within the reaction cavity330leaking to the second package350.

In one preferred embodiment of the present invention, the first package340comprises a pit fabricated within the plate structure310and a covering covered thereonto. In addition, the first channel370and the second channel380are both sealed by stoppers. When the covering of the first package340is pressed, the recognizing molecule contained therein breaks through the stopper and flows into the reaction cavity330. When the second package350is pressurized, the buffer solution contained therein breaks through the stopper and spills into the reaction cavity330. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

As described in Example 1, the immunoassay cartridge300in this example further comprises a removable lid390capable of being covered onto the plate structure310. On the inner surface of the removable lid390is lined with an adsorption pad395. In one preferred embodiment of the present invention, the adsorption pad395is made of adsorbing materials like membranes, tissues, or polymers such as starch-acrylic acid graft copolymer, copoly (vinyl alchol-acrylic acid), cross-linking copolyacrylic acid or combinations thereof. It is appreciated that these are not used to limit the score and the spirit of the present invention.

When the immunoassay cartridge300is accommodated by an analyzer (not shown herein), the biological sample flowing into the reaction cavity330through the sample receiving end360reacts with the analyte-binding molecule immobilized in the reaction cavity330, thus forming a complex of the analyte and the analyte-binding molecule.

Thereafter, the removable lid390is covered onto the plate structure310to allow the adsorption pad395attached thereinside to adsorb the liquid in the reaction cavity330. Next, the removable lid390is removed from the plate structure310.

It is noted that the covering of the first package340should be avoided swelling out of the upper surface3101of the plate structure310when the first package340is loaded with the recognizing molecule. Accordingly, the recognizing molecule contained in the first package340will not be squeezed into the reaction cavity330while covering the removable lid390onto the plate structure310.

After forming the complex of the analyte and the analyte-binding molecule, the second package350is automatically compressed to have the buffer solution therein break the stopper and spill into the reaction cavity330via the second channel380for wash. Next, the used adsorption pad395should be replaced with a fresh one by the analyzer for avoiding contaminations. That is, the used adsorption pad395should be replaced with a fresh one every time when it is used to adsorb the liquid within the reaction cavity330.

After that, the removable lid390is covered to the plate structure310to allow the new adsorption pad395attached thereinside to adsorb the liquid in the reaction cavity330.

In another embodiment, the adsorption pad395is directly installed in the analyzer instead of being attached on the removable lid390. The purpose of the adsorption pad395is to adsorb the liquid within the reaction cavity330rapidly. Accordingly, various possible modifications and substitutions, without departing from the purpose, should be should be included in the present invention. After evacuating the liquid within the reaction cavity330using the adsorption pad395, the removable lid390is removed from the plate structure310.

Next, the recognizing molecule such as a gold-conjugated antibody in the first package340is automatically squeezed to break the stopper and flow into the reaction cavity330via the first channel370, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid390is covered onto the plate structure310to allow the adsorption pad395attached thereinside to adsorb the liquid contained in the reaction cavity330. The removable lid390is then removed from the plate structure310. Lastly, the second package350is automatically compressed again to force the buffer solution to spill into the reaction cavity330for rinse.

It is noted that, in another embodiment, diameters of the first channel370and the second channel380are designed to allow the recognizing molecule and the buffer solution to stay in the first package340and the second package350and then flow into the reaction cavity330when the first package340and the second package350suffer the pressure. That is, the stopper described above is absent in this embodiment.

In one preferred embodiment of the present invention, the third package397comprises a pit made within the plate structure330and a covering covered onto the pit. In another embodiment, the third package397comprises a container coupled to the plate structure330and a covering covered onto the container.

In the enzymatic immunoassay, the complex of the analyte and the analyte-binding molecule is bound with an enzyme. The enzyme-bound complex is then contacted with the signal molecule, e.g. a substrate, for the enzyme to produce a detectable substance.

As described above, a third channel398communicates the third package397and the reaction cavity330. Accordingly, the third package397is adjacent to the reaction cavity330.

In one preferred embodiment of the present invention, the third package397comprises a pit fabricated within the plate structure310and a covering covered onto the pit. Further, the third channel398is sealed by a stopper. When the covering of the third package397is pressurized, the signal molecule inside the third package397breaks through the stopper and flows into the reaction cavity330. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

After forming the first product of the complex and the recognizing molecule and washing the reaction cavity330with the buffer solution, the signal molecule in the third package397is automatically pressed to break the stopper and flow into the reaction cavity330via the third channel398, thus forming a second product of the first product and the signal molecule. Subsequently, the removable lid390is covered to the plate structure310to allow the adsorption pad395attached thereinside to adsorb the liquid in the reaction cavity330. Then, the removable lid390is removed from the plate structure310.

Lastly, the second package350is automatically pressed again to force the buffer solution to flow into the reaction cavity330for rinse. The second product is subjected to detect the concentration of the analyte in the biological sample.

It is noted that, in another embodiment, a diameter of the third channel398is designed to allow the signal molecule to stay in the third package397and then flow into the reaction cavity330when the third package397is pressed. In other words, the stopper described above is absent herein.

Referring toFIG. 4, a side view of an immunoassay cartridge400is shown herein. The immunoassay cartridge400includes a supporting plate410. A sample loading chamber420is fabricated within the supporting plate410and provided for receiving and containing a biological sample suspected of an analyte. In one preferred embodiment of the present invention, the biological sample comprises a blood sample, a serum sample or a saliva sample.

It is noted that the sample loading chamber420can be omitted optionally according to practical needs. In this case, the biological sample is loaded in the reaction cavity430directly.

Within the supporting plate410also includes a reaction cavity430. At least one analyte-binding molecule is immobilized on the bottom surface of the reaction cavity430. In one preferred embodiment of the present invention, the analyte-binding molecule(s) in the reaction cavity430is/are printed to from an array. The method of immobilizing the analyte-binding molecule within the reaction cavity430comprises physical adsorption, cross-linking, covalent binding, entrapment or any combination thereof.

In one preferred embodiment of the present invention, the analyte is an antigen and the analyte-binding molecule is a first antibody correspondingly. In another embodiment, the analyte-binding molecule is a ligand when the analyte is a protein.

Still referring toFIG. 4, a first channel460connects the sample loading chamber420and the reaction cavity430to allow the biological sample in the sample loading chamber420to flow into the reaction cavity430.

The immunoassay cartridge400further comprises a first package440and a second package450filled with a recognizing molecule and a signal molecule, respectively. It is noted that the recognizing molecule is preferably a second antibody such as a gold-conjugated antibody, and the signal molecule is preferably a substrate while the analyte is the antigen and the analyte-binding molecule is the first antibody. However, it is appreciated that these are not used to limit the score and the spirit of the present invention.

As shown in the figure, a second channel470is used to communicate the first package440and the reaction cavity430, whereas the third channel480is used to communicate the second package450and the reaction cavity430. The supporting plate410is covered onto a tank455full of a buffer solution to form an enclosure. A conduit456connects the tank455and the reaction cavity430for allowing the buffer solution in the tank455to spill into the reaction cavity430.

In one preferred embodiment of the present invention, the first package440and the second package450comprise pits fabricated within the supporting plate410and coverings covered onto the pits. In another embodiment, the first package440and the second package450comprise containers coupled to the enclosure and coverings covered onto the containers.

The second channel470and the third channel480are both sealed by stoppers. When the covering of the first package440or the second package450is pressed, the recognizing molecule or the substrate inside the respective first package440and the second package450breaks through the stopper and flows into the reaction cavity430. In the embodiment, the covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

In another embodiment, diameters of the second channel470and the third channel480are designed to allow the recognizing molecule and the substrate to stay in the first package440and the second package450and then flow into the reaction cavity430when they are pressurized. That is, the stopper described above is absent in this embodiment.

It is noted that the first channel460, the second channel470and the third channel480are not used to limit the score and the spirit of the present invention. That is, the first channel460, the second channel470and the third channel480can be replaced with pipes or the like.

The immunoassay cartridge400further comprises a removable lid490capable of being covered onto the supporting plate410. On the inner surface of the removable lid490is lined with an adsorption pad495. In one preferred embodiment of the present invention, the adsorption pad495is made of adsorbing materials like membranes, tissues, or polymers such as starch-acrylic acid graft copolymer, copoly (vinyl alchol-acrylic acid), cross-linking copolyacrylic acid or combinations thereof. It is appreciated that these are not used to limit the score and the spirit of the present invention.

When the immunoassay cartridge400is accommodated by an analyzer (not shown herein), the biological sample flowing into the reaction cavity430through the first channel460reacts with the analyte-binding molecule immobilized in the reaction cavity430, thus forming a complex of the analyte and the analyte-binding molecule.

Thereafter, the removable lid490is covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Next, the removable lid490is removed from the supporting plate410.

It is noted that the covering of the first package440should be avoided swelling out of the upper surface4101of the supporting plate410when the first package440is loaded with the recognizing molecule. Accordingly, the recognizing molecule contained in the first package440will not be squeezed into the reaction cavity430while covering the removable lid490onto the plate structure310.

After forming the complex of the analyte and the analyte-binding molecule, the buffer solution in the tank455is pressurized to spill into the reaction cavity430via the conduit456for wash. In the example, the buffer solution in the tank455is pumped to the reaction cavity430via the conduit456.

Next, the used adsorption pad495should be replaced with a fresh one by the analyzer for avoiding contaminations. That is, the used adsorption pad495should be replaced with a fresh one every time when it is used to adsorb the liquid within the reaction cavity430.

Thereafter, the removable lid490is covered to the supporting plate410to allow the new adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430.

In another embodiment, the adsorption pad495is directly installed in the analyzer instead of being attached on the removable lid490. The purpose of the adsorption pad495is to adsorb the liquid within the reaction cavity430rapidly. Accordingly, various possible modifications and substitutions, without departing from the purpose, should be should be included in the present invention. After evacuating the liquid within the reaction cavity430using the adsorption pad495, the removable lid490is removed from the supporting plate410.

Next, the recognizing molecule such as a gold-conjugated antibody in the first package440is automatically squeezed to break the stopper and flow into the reaction cavity430via the second channel470, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid490is covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid contained in the reaction cavity430. The removable lid490is then removed from the plate structure410.

After forming the first product of the complex and the recognizing molecule, the buffer solution in the tank455is automatically pressurized to spill into the reaction cavity430via the conduit456for rinse. Then, the removable lid490is covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Thereafter, the removable lid490is removed from the supporting plate410.

Next, the signal molecule in the second package450is automatically squeezed to break the stopper and flow into the reaction cavity430via the third channel480, thus forming a second product of the intermediate and the signal molecule. After that, the removable lid490is covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid contained in the reaction cavity430. The removable lid490is then removed from the plate structure410.

After forming the second product, the buffer solution in the tank455is pressurized to spill into the reaction cavity430via the conduit456for rinse. Then, the removable lid490is covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Lastly, the removable lid490is removed from the supporting plate410.

Referring toFIG. 5A, a side view of an immunoassay cartridge500is shown herein. The immunoassay cartridge500includes a plate structure510and an extending portion520coupled thereto. In an embodiment of the present invention, the extending portion520and the plate structure510are made by a procedure of one-piece molding. In another embodiment, the extending portion520and the plate structure510are fabricated separately and then assembled together.

Within the plate structure510includes a reaction cavity530, and at least one analyte-binding molecule is immobilized on the bottom surface of the reaction cavity530. In one preferred embodiment of the present invention, the analyte-binding molecule(s) within the reaction cavity530is/are printed to form an array. In addition, the method of immobilizing the analyte-binding molecule in the reaction cavity530comprises physical adsorption, cross-linking, covalent binding, entrapment or any combination thereof.

One end561of a sample receiving end560is connected to the reaction cavity530. The other end562of the sample receiving end560penetrates through the extending portion520and has an opening563on a sidewall5201of the extending portion520. In this embodiment, the extending portion520is provided to protect the sample receiving end560from being broken.

A biological sample suspected of an analyte is received from the opening563and flows into the reaction cavity530via the sample receiving end560. In another embodiment, the biological sample is loaded into the reaction cavity530. That is, the sample receiving end560can be optionally omitted from the immunoassay cartridge500according to practical needs.

In a preferred embodiment of the present invention, the biological sample comprises a blood sample, a serum sample or a saliva sample. In addition, the sample receiving end560is a capillary.

In one preferred embodiment of the present invention, the analyte is an antigen and the analyte-binding molecule is a first antibody correspondingly. In another embodiment, the analyte-binding molecule is a ligand when the analyte is a protein.

The immunoassay cartridge500further comprises a first package540and a second package550filled with a recognizing molecule and a buffer solution, respectively. It is noted that the recognizing molecule is preferably a second antibody when the analyte is the antigen and the analyte-binding molecule is the first antibody. However, it is appreciated that these are not used to limit the score and the spirit of the present invention. One of differences between Example 5 and Example 3 is that the first package540in this example is located under the plate structure510.

A first channel570within the plate structure510communicates the first package540and reaction cavity530, whereas a second channel580connects the second package550and reaction cavity530.

In one preferred embodiment of the present invention, the first channel570and the second channel580are both sealed by stoppers. When the first package540suffers the pressure, the recognizing molecule contained therein breaks through the stopper and spills into the reaction cavity530. When the second package550suffers the pressure, the buffer solution contained therein breaks through the stopper and spills into the reaction cavity530.

As described in Example 1, the immunoassay cartridge500further comprises a removable lid590capable of being covered onto the plate structure510. On the inner surface of the removable lid590is lined with an adsorption pad595. In one preferred embodiment of the present invention, the adsorption pad595is made of adsorbing materials like membranes, tissues, or polymers such as starch-acrylic acid graft copolymer, copoly (vinyl alchol-acrylic acid), cross-linking copolyacrylic acid or combinations thereof. It is appreciated that these are not used to limit the score and the spirit of the present invention.

When the immunoassay cartridge500is accommodated by an analyzer (not shown herein), the biological sample flowing into the reaction cavity530through the sample receiving end560reacts with the analyte-binding molecule immobilized in the reaction cavity530, thus forming a complex of the analyte and the analyte-binding molecule.

Thereafter, the removable lid590is covered onto the plate structure510to allow the adsorption pad595attached thereinside to adsorb the liquid in the reaction cavity530. Next, the removable lid590is removed from the plate structure510.

After forming the complex of the analyte and the analyte-binding molecule, the buffer solution in the second package550is forced to break the stopper and spill into the reaction cavity530via the second channel580for wash.

Next, the used adsorption pad595should be replaced with a fresh one by the analyzer for avoiding contaminations. That is, the used adsorption pad595should be replaced with a fresh one every time when it is used to adsorb the liquid within the reaction cavity530.

Thereafter, the removable lid590is covered to the plate structure510to allow the new adsorption pad595attached thereinside to adsorb the liquid in the reaction cavity530.

In another embodiment, the adsorption pad595is directly installed in the analyzer instead of being attached on the removable lid590. The purpose of the adsorption pad595is to adsorb the liquid within the reaction cavity530rapidly. Accordingly, various possible modifications and substitutions, without departing from the purpose, should be should be included in the present invention. After evacuating the liquid within the reaction cavity530using the adsorption pad595, the removable lid590is removed from the plate structure510.

Next, the first package540is automatically compressed to have the recognizing molecule, such as a gold-conjugated antibody, contained therein break the stopper and flow into the reaction cavity530via the first channel570, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid590is covered onto the plate structure510to allow the adsorption pad595attached thereinside to adsorb the liquid contained in the reaction cavity530. The removable lid590is then removed from the plate structure510.

Lastly, the second package550is automatically compressed again to force the buffer solution to spill into the reaction cavity530for rinse.

It is noted that, in another embodiment, a diameter of the first channel570is designed to allow the recognizing molecule to stay in the first package540and then spill into the reaction cavity530when the first package540is pressurized. A diameter of the second channel580is designed to allow the buffer solution to spill into the reaction cavity530when the second package550is pressurized. In other words, the stopper described above is absent in this embodiment.

Furthermore, the diameters of the first channel570and the second channel580are designed to avoid the liquid within the reaction cavity530leaking to the first package540or the second package550.

In one preferred embodiment of the present invention, the third package597comprises a pit made within the plate structure530and a covering covered onto the pit. In another embodiment, the third package597comprises a container coupled to the plate structure530and a covering covered onto the container.

In the enzymatic immunoassay, the complex of the analyte and the analyte-binding molecule is bound with an enzyme. The enzyme-bound complex is then contacted with the signal molecule, e.g. a substrate, for the enzyme to produce a detectable substance.

As described above, a third channel598communicates the third package597and the reaction cavity530. Accordingly, the third package597is adjacent to the reaction cavity530.

In one preferred embodiment of the present invention, the third package597comprises a pit fabricated within the plate structure510and a covering covered onto the pit. Further, the third channel598is sealed by a stopper. When the covering of the third package597is pressurized, the signal molecule inside the third package597breaks through the stopper and flows into the reaction cavity530. The covering is preferably made of polymer such as PDMS or PET (polyethylene terephthalate). The stopper is made of a wide variety of materials including silicon, polyvinyl alcohol (PVA), polystyrene (PS) or other polymers.

After forming the first product of the complex and the recognizing molecule, the signal molecule in the third package597is automatically pressed to break the stopper and flow into the reaction cavity530via the third channel598, thus forming a second product of the first product and the signal molecule. Subsequently, the removable lid590is covered to the plate structure510to allow the adsorption pad595attached thereinside to adsorb the liquid in the reaction cavity530. Then, the removable lid590is removed from the plate structure510.

Lastly, the second package550is automatically pressed again to force the buffer solution to flow into the reaction cavity530for rinse. The second product is subjected to detect the concentration of the analyte in the biological sample.

It is noted that, in another embodiment, a diameter of the third channel598is designed to allow the signal molecule to stay in the third package597and then flow into the reaction cavity530when the third package597is pressed. In other words, the stopper described above is absent herein.

Referring toFIG. 5B, the third package597is located under the plate structure510. A third channel598communicating the third package597and reaction cavity530is sealed by a stopper. When the third package597is compressed, the signal molecule contained therein breaks through the stopper and spills into the reaction cavity530, thus forming the second product of the first product and the signal molecule as described above.

In this case, a diameter of the third channel598is designed to avoid the liquid within the reaction cavity530leaking to the third package597.

Below, carcinoembryonic antigen (CEA) used in the immunoassay cartridge400of Example 4 is illustrated to elucidate how the immunoassay of the present invention works. It is appreciated that, however, the CEA system exemplified herein is not used to limit the score and the spirit of the present invention.

CEA, a 180 kD intercellular adhesion molecule expressed in high concentrations in the fetus but normally not found in adult serum because the synthesis of this protein ceases after birth. However reappear in a high concentration in the sera of patients with colorectal (57%), gastric (41%), hepatocellular (45%), pancreatic (59%) and biliary (59%) carcinoma. The serum concentration of CEA can also be elevated in benign diseases of the colorectum (inflammatory bowel disease 17%), stomach (chronic gastritis and peptic ulcer 14%), liver (cirrhosis and hepatitis 17%) and pancreas (21%). Elevated levels of CEA have also been observed in patients with inflammatory nonmalignant diseases like pulmonary emphysema, alcoholic cirrhosis, and pancreatitis and in heavy smokers. In contrast to cancer these elevations are transitory. The serum levels drop back into the normal range within a few weeks.

Before carrying out the immunoassay with the immunoassay cartridge, a biological sample suspected of the CEA is loaded into the reaction cavity430.

When the immunoassay cartridge400is accommodated by the analyzer (not shown herein), the biological sample suspected of CEA flows into the reaction cavity430through the first channel460and reacts with a capture antibody, e.g. mouse anti-human CEA monoclonal antibody, immobilized in the reaction cavity430. Hence, a complex of the CEA and the capture antibody is formed.

Thereafter, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Next, the removable lid490is automatically removed from the supporting plate410.

After forming the complex of the of the CEA and the capture antibody, the buffer solution, preferably 0.1 M phosphate buffer at pH 7.4, in the tank455is pumped to the reaction cavity430via the conduit456for rinse.

Thereafter, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Then, the removable lid490is automatically removed from the supporting plate410.

Next, the recognizing molecule, e.g. immunoglobulin (IgG) fraction of rabbit antiserum to human CEA, in the first package440is squeezed to break the stopper and flow into the reaction cavity430via the second channel470, thus forming a first product of the complex and the recognizing molecule. After that, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid contained in the reaction cavity430. The removable lid490is then automatically removed from the plate structure410.

After forming the first product of the complex and the recognizing molecule, the buffer solution in the tank455is pumped to the reaction cavity430via the conduit456for rinse. Then, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Thereafter, the removable lid490is automatically removed from the supporting plate410.

The detection process may be performed if the recognizing molecule has already conjugated with signal molecule(s) such as fluorophore(s), isotope(s), or gold particle(s).

Accordingly, the recognizing molecule, such as sheep anti-rabbit IgG antibody that conjugated with the signal molecule(s), is contained in the second package450and is squeezed to break the stopper and flow into the reaction cavity430via the third channel480, thus forming a second product of the first product and the recognizing molecule (in other words, the first package440is absent in this embodiment). After that, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid contained in the reaction cavity430. The removable lid490is then automatically removed from the plate structure410.

After forming the product, the buffer solution in the tank455is pumped to the reaction cavity430via the conduit456for rinse. Then, the removable lid490is automatically covered onto the supporting plate410to allow the adsorption pad495attached thereinside to adsorb the liquid in the reaction cavity430. Lastly, the removable lid490is automatically removed from the supporting plate410.

While the preferred embodiment of the invention has been illustrated and described, it is appreciated that various modifications, additions and substitutions can be made therein without departing from the spirit and scope of the invention.