JP2012247188A - Clinical examination using nanocarbon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection method in which, in a clinical examination using the substance originating from a living thing such as blood, as a sample, only a component to be detected can be detected with high sensitivity by being kept as it is without previously removing a biological material such as a corpuscle component other than an object to be detected.SOLUTION: A nanocarbon such as a single-walled carbon nanotube (SWCNT) emits near infrared light by photoexcitation and such emission is utilized, the near infrared light emitted by the detected object is utilized for detection by labeling a detection probe by the nanocarbon, thereby capable of detecting only a component to be detected with high sensitivity by being kept as it is without previously removing a biological material such as a corpuscle component other than an object to be detected.

Description

  The present invention relates to a clinical examination technique using a labeled probe that emits light in the near infrared region.

Searches for biomarkers of various diseases are being promoted against the background of recent technological development, and candidate molecules that are likely to be useful for early detection of diseases such as “cancer” have been reported one after another. Ikehara et al., One of the inventors of the present invention, at the Glycotechnology Research Center of the National Institute of Advanced Industrial Science and Technology makes the most of the advantages of glycan analysis technology, and it appears that it appears as the disease develops and progresses We have been conducting research using the glycoprotein (Fig. 1) (= glycosylated isomer) as a disease biomarker. By detecting disease markers such as unfinished glycoproteins, cancer and lifestyle-related diseases can be detected early.
Recently, Ikehara et al. Discovered an incomplete glycan biomarker that can measure fibrosis caused by hepatitis and enabled detection evaluation in blood tests instead of highly invasive biopsy. Furthermore, we have succeeded in discovering unfinished glycan biomarkers that can be evaluated for the recurrence of gastric cancer in the abdominal cavity and the presence and progression of extremely difficult tumors such as bile duct cancer and ovarian cancer. In the future, if development is carried out using the same approach, it is expected to be applicable to laboratory diagnosis of arteriosclerosis and Alzheimer's disease. However, detection techniques commonly used in current clinical tests are not sufficient to achieve ultra-early diagnosis of these diseases by detecting disease marker molecules present in blood, body cavity fluid, bile, etc. It is difficult to obtain sensitivity.

  In general, in a clinical test using blood, body cavity fluid, bile, etc. as a sample, cell components such as blood cells (red blood cells, white blood cells, platelets, mesothelial cells, dropped cells) are removed by centrifugation or filter filtration. However, since it cannot be removed by any operation, not a few of these biological substances are mixed, and the light of the probe molecules used for detecting the disease biomarker is disturbed and absorbed, and the disease detection sensitivity is lowered. At the same time, cells such as blood cell components, and proteins contained in body cavity fluid and bile emit nonspecifically, which causes noise when detecting probe luminescence. Interference absorption and self-emission noise of probe luminescence due to such components hinders high-sensitivity detection of various disease biomarkers for the purpose of ultra-early diagnosis, and are very serious obstacles to their practical application.

Journal of the American Chemical Society (2007) 5612-5620. Analytical Chemistry (2009) Volume 81, 7885-7891. Leeuw et al. Nano Lett. 2007,7 (9), 2650-4

  The present invention is highly sensitive to only a detection target component without removing in advance a biological material such as blood cells and components other than the detection target in a clinical test using a biological derived material such as blood, body cavity fluid, and bile as a sample. It is an object of the present invention to provide a detection method that can be detected by the above method.

  The present inventors have focused on the fact that nanocarbons such as single-walled carbon nanotubes (SWCNT) emit near-infrared light by photoexcitation. By labeling the detection probe with nanocarbon, the detected object emits near-infrared light. It has been found that by using infrared light for detection, it is possible to detect only the detection target component with high sensitivity without removing biological substances such as blood cell components other than the detection target from the sample in advance. The present invention has been completed.

Since near-infrared light has high biological permeability, technological development has been attempted with the goal of increasing the sensitivity of in-vivo diagnostics such as in vivo imaging and axillary lymph node examination. Even in clinical examinations that search for blood and body cavity fluids, if near-infrared light can be used, high sensitivity and simultaneous detection of many types of disease markers are possible, but this has not been realized. For realization, a probe that efficiently emits light in the near-infrared wavelength region is required.
SWCNT emits light at 700-2300 nm by photoexcitation at 450-1350 nm (FIG. 2 shows a light emission map of a part of the wavelength range). This wavelength range includes a wavelength range (wavelength 600 to 1300 nm) that is difficult to be absorbed by a biological substance or has little self-emission. SWCNT has an optical durability of 10 times or more compared to near-infrared dye molecules and near-infrared quantum dots, and can be chemically modified. In other words, SWCNT has an unparalleled superiority as a near-infrared light emitting probe, and is a powerful new material that enables the realization of next-generation clinical tests using near-infrared light. SWCNT emission was discovered in 2002, and in vivo SWCNT imaging has been attempted (Non-Patent Document 3), but the most promising application to clinical testing has not been done so far. .

In order to detect a very small amount of detection target in a sample in which impurities derived from a living body exist by emitting near-infrared light, an apparatus that detects weak near-infrared light is also required.
For detection of near-infrared light with a wavelength of 1000 to 2000 nm, an InGaAs photodiode array connected to a silicon charge integrating amplifier is usually used. However, near room temperature, the photodiode has a large dark current and is allowed by a silicon IC. With capacitance, sufficient integration time cannot be secured. Although this point can be solved by cooling the photodiode with liquid nitrogen, it is difficult to put it into practical use in a clinical laboratory in view of problems of price and operability. On the other hand, Ogura, one of the present inventors, previously developed a heterobipolar phototransistor provided with a surface current blocking layer so as to enable measurement of extremely weak light near room temperature (PCT / JP2009). / 067689 patent pending).
As a feature of this element, since the internal amplification function is large (current amplification factor β to several thousand), it is difficult to be affected by noise from an external electric circuit, and has the effect of accumulating photoexcited carriers in the base region. It is built-in, and has sensitivity in a wide wavelength range from visible to infrared.
Furthermore, in the case of fluorescence or transmission spectrum measurement, it is possible to remove the dark current component by modulating the light source and synchronously detecting the detection system (lock-in detection). The circuit needs to be integrated into the IC, and sufficient measures have not been taken.
By connecting a lock-in amplifier array using a silicon IC to each element of the photodiode array, the present inventors made femtowatt level faint light detection possible even in a room temperature array (the above-mentioned PCT / JP2009 / 067689).

  That is, in the present invention, in the presence of the fundamental technology of system equipment that can detect near infrared light with high sensitivity, nanocarbon such as SWCNT that emits light with near infrared light is used as a labeling substrate to detect a target object. By modifying necessary probes such as antibodies, in addition to the conventional visible wavelength band, it is possible to perform a specimen test of a blood biochemical test using a fluorescent label in the near infrared wavelength band. Furthermore, by extending the detection wavelength band to 2 μm, it becomes possible to perform HbA1c quantification and blood glucose level measurement in parallel with the light absorption and transmission characteristics of glycated hemoglobin and glucose in erythrocytes, thereby preventing adult diseases. Contributes greatly to screening.

Specifically, the present application provides the following inventions.
<1> using the unique optical properties (light absorption, light emission, Raman scattering light) of nanocarbon (single-walled carbon nanotube, multi-walled carbon nanotube, carbon nanohorn, nanographene, other nanocarbon), Immunoassay method or clinical test method.
<2> The immunoassay method or clinical test method according to <1>, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by photoexcitation at wavelengths of 450-1350 nm are used as a luminescent label. .
<3> A luminescent labeling substance containing nanocarbon (single-walled carbon nanotube, multi-walled carbon nanotube, carbon nanohorn, nanographene, and other nanocarbon).
<4> The luminescent labeling substance according to <3>, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 70-2300 nm by photoexcitation at a wavelength of 450-1350 nm.
<5> Nanoparticles bonded to an antibody that recognizes a specific antigen, and the antigen-antibody reaction is detected by measuring the fluorescence, Raman light, and light absorption of the nanocarbon, <3> or <4 The luminescent labeling substance according to>.
<6> Streptavidin and related molecules of streptavidin are combined with nanocarbon, and the reaction product of biotinylated antibody and streptavidin bound to the antigen is detected by measuring the fluorescence, Raman light, and light absorption of nanocarbon. The luminescent labeling substance according to <3> or <4>, wherein
<7> Combining nanocarbon with immunoglobulin-binding protein such as Protein G, Protein A, etc. In order to detect the antibody bound to the antigen, the binding reaction between the antibody and the antibody-binding protein is carried out by fluorescence of nanocarbon, Raman light, The luminescent labeling substance according to <3> or <4>, which is detected by measuring light absorption.
<8> Various antigens (including cancer-related antigens such as proteins and sugar chains, and disease biomarkers) present in blood and body cavity fluids when a specific antigen becomes normal or pathological The luminescent labeling substance according to <5>, which is characterized in that it exists.
<9> For samples enriched using antibodies against various antigens bound to carriers such as magnetic beads, Sepharose, Sephadex, etc., detect the antigens by nano-carbon near-infrared emission measurement after binding the nanocarbon-labeled antibodies. The immunoassay method or clinical test method according to <1> or <2>.
<10> An antigen that is a disease marker is labeled with a nanocarbon-labeled antibody, collected by electrophoresis, and then detected by near-infrared emission measurement of nanocarbon. <1> or <1> The immunoassay method or clinical test method according to 2>.
<11> Detecting a plurality of antigens in different emission wavelength bands by combining the luminescent clinical test method according to <1> to <10> or a combination with another luminescent clinical test method in a single specimen. An immunoassay method or a clinical test method characterized by
<12> The immunoassay method or clinical test method according to <11>, wherein, in a single specimen, a ratio of emission intensity from a plurality of antigens in different emission wavelength bands is used.
<13> Clinical examination for modulating near-infrared emission by modulating single or plural excitation light in the ultraviolet, visible, near-infrared and infrared emission measurement of <11> or <12> apparatus.
<14> An immunoassay characterized by using a SWCNT-labeled antibody as a secondary antibody in place of peroxidase or alkaline phosphatase used as a secondary antibody in an enzyme immunoassay (ELISA).
<15> A measurement method for measuring a plurality of antibody reactions in the same specimen by using a fluorescent label at another emission wavelength in addition to the emission wavelength of SWCNT described in <2>.

According to the present invention, by labeling a detection target with nanocarbon that emits near-infrared light, biological substances such as blood cell components other than the detection target are removed in advance in a clinical test using a biological derived material such as blood as a sample. Instead, only the detection target component can be detected with high sensitivity.
With flow cytometers and confocal laser microscopes, the amount of information that can be obtained has increased dramatically due to the expansion of the usable wavelength range. According to the present invention, the clinical laboratory equipment can be greatly expanded from the conventional wavelength range of 400-500 nm to 400-2300 nm and the near infrared region, and the obtained information is further increased. For example, by combining different excitation luminescence probes, several kinds of biomarkers can be detected and evaluated simultaneously in one assay. According to the present invention, the use of near-infrared light that is highly permeable to biological substances enables examination of biological samples derived from living organisms without separating them into serum or plasma. The information that was lost can be obtained. Moreover, even during serum testing, the biomarker-derived signal light absorption and non-specific noise due to the mixed blood cells and platelet components can be suppressed, making it possible to detect trace biomarkers, and extremely early such as cancer and Alzheimer's disease. It can greatly contribute to the development of medicine and medical care by enabling discovery and early treatment.
The clinical test system using near infrared emission of SWCNT of the present invention will bring near infrared light to clinical tests for the first time in the world, and will bring revolutionary progress to the diagnostics and diagnostic equipment industries.

Comparison schematic diagram of completed glycoprotein and unfinished product. SWCNT two-dimensional emission map. Conceptual diagram of detection of disease markers (antigens such as incomplete glycoproteins) using SWCNT. The molecular structure of DSPE-PEG. Molecular structure of NHS-DSPE-PEG. Infrared antibody reaction detector The cross-sectional schematic diagram of the whole blood spectroscopic fluorescence apparatus equipped with the small near-infrared spectrometer. FIG. 8 is a comparison diagram of a whole blood fluorescence analysis spectrum by the apparatus of FIG. 7 and a spectrum by a conventional method. An example of an apparatus using a light emitting diode, a filter, and a light receiving element instead of a small spectroscope. 1 is a schematic diagram of an automatic clinical laboratory test apparatus according to the present invention.

Example 1. Detection of antigen-antibody reaction by near-infrared emission using SWCNT 1) In this example, an antigen-antibody reaction was performed using ovalbumin (OVA) as a model antigen, and the antigen-antibody reaction was detected by near-infrared emission.
First, a monoclonal antibody against a model antigen was prepared and immobilized on an ELISA plate. The sample solution containing the model antigen was allowed to react at room temperature for 2 hours on a plate on which the model antigen was immobilized, and then the supernatant was discarded and washed thoroughly with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT was reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. Washing and removing unreacted SWCNT-labeled antibody by washing the plate thoroughly with phosphate buffer or Tris buffer, and detecting near-infrared fluorescence derived from SWCNT detected on the plate. Detection was performed.
2) In addition to the system using the antibody immobilized on the ELISA plate, the antibody can be bound to Protein G beads and used in batch processing. When Protein G beads are used, the antibody can be bound to the beads via binding between Protein G bound to the beads and the Fc part of the antibody. The antibody-immobilized beads were reacted in a sample solution containing a model antigen at room temperature for 2 hours, and then the supernatant was discarded and washed thoroughly with phosphate buffer or Tris buffer. Next, a polyclonal antibody probe (SWCNT-labeled antibody) against the model antigen labeled with SWCNT was reacted in a phosphate buffer or Tris buffer at room temperature for 2 hours. By washing the beads thoroughly, unreacted SWCNT-labeled antibodies were removed, and by detecting near-infrared fluorescence derived from SWCNTs detected on the beads, the antigen-antibody reaction was detected.

The preparation of SWCNT-labeled antibodies used in these antigen-antibody reactions was performed according to the following procedure.
Binding of DSPE-PEG and antibody 1) Dissolve 125 μg of antibody in 6 μl of 50 mM phosphate buffer (pH 7.2), add 5 μg of DSPE-PEG (see FIG. 4, terminal amino group) and stir. Next, 8 μg of WSC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) catalyst is added and stirred with a stirrer at 4 ° C. for 10 hours. Filter through a filter, and collect the reactant remaining on the filter by dissolving it in a buffer.
Or
2) Dissolve 125 μg of antibody in 2.5 μl of NaHCO ₃ buffer. NHS-PEG-DSPE (see FIG. 5) is dissolved in 5 μg of pure water and added to the antibody solution. Stir at 4 ° C. for 6 hours using a vortex to react. The reaction solution is purified by gel filtration and collected.
Treatment of SWCNT First, about 1 mg of carbon nanotube (SWCNT) was immersed in about 20 ml of 1 wt% sodium dodecylbenzenesulfonate (SDBS) aqueous solution and dispersed by an ultrasonic crusher. The obtained dispersion was ultracentrifuged at 171000 g for 2.5 hours to obtain a translucent supernatant (about 10 ml).
Preparation of SWCNT- labeled antibody probe Add 1 mg of DSPE-PEG to which antibody is bound per 1 ml of the supernatant of this SWCNT dispersion, and apply it to a bath-type ultrasonic cleaner for 3 minutes to completely dissolve the antibody-bound DSPE-PEG. It was. Then, it put into the dialysis membrane which lets the molecule | numerator of molecular weight 3500 or less pass, and dialyzed for 5 days. This dialysis operation replaced SDBS with antibody-bound DSPE-PEG.

The detailed procedure of the antigen-antibody reaction is as follows.
Reaction system measurement on ELISA plate
The plate is coated for 2 hours at room temperature using 50 μl of anti-OVA antibody solution adjusted to 20 μg / ml with PBS or 100 mM bicarbonate / carbonate coating buffer. After thoroughly washing with PBS, 200 μl of 5% BSA solution dissolved in PBS is put into a well and blocked at room temperature for 30 minutes. After thoroughly washing with PBS, add the sample, react at room temperature for 2 hours, and then wash well with PBS. Next, the SWCNT-antibody probe diluted to 20 μg / ml with PBS is reacted at room temperature for 2 hours, and then washed well with PBS.
After the plate is dried, luminescence is detected.
Reaction system measurement in batch processing using magnetic beads
According to the protocol of Dynabeads Protein G, 200 μl of anti-OVA antibody adjusted to 5 μg / ml with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes to prepare Dynabeads to which the antibody was bound. Next, 500 μl of a sample solution containing a model antigen was reacted at room temperature for 2 hours, and the antigen was recovered with Dynabeads to which the antibody was bound.
Next, after thoroughly washing with PBS, the mixture is reacted with 100 μl of SWCNT-antibody probe diluted to 20 μg / ml with PBS for 2 hours at room temperature, and then thoroughly washed with PBS.
After removing Buffer, SWCNT luminescence is detected.

In addition, detection of luminescence from the labeled probe is performed as follows.
By irradiating magnetic beads-antigen-SWCNT-DSPE-PEG-antibody with light having a detection wavelength of 660 nm, light emission at a wavelength of about 1200 nm can be observed from SWCNT. Its near-infrared emission spectrum is measured by a spectroscopic system equipped with an InGaAs detector.
Furthermore, in the case of fluorescence measurement, since excitation light can be modulated, the detection level of weak light is set to a dark current equivalent level (using a lock-in amplifier detection method that performs synchronous detection corresponding to the modulation period ( It is possible to improve from a few pW) to a shot noise limit (up to several tens of fW). Fig. 6 shows a high-sensitivity infrared light detection array module in which a lock-in amplifier and an AD converter are connected to each cell of an InGaAs near-infrared phototransistor array having an internal amplification action. Magnetic beads-antigen-SWCNT-DSPE-PEG- The example used for the detection of an antibody is shown. In this embodiment, since the influence of dark current can be suppressed, faint watt level faint light can be detected without using a cryogen.

Example 2.3 Production of a device for detecting modified isomers by wavelength In clinical tests, the degree of post-translational modification of proteins present in serum is measured to evaluate the presence and progression of disease. And you have important information to decide the treatment policy. Examples include 1) quantitative detection of glycated proteins represented by glycated hemoglobin, and 2) sugars modified with characteristic sugar chain structures that appear in connection with diseases such as fetal cancer antigen (AFP) and MUC1. Mention can be made of quantitative detection of proteins. 1) Glycated protein is a substance resulting from the non-enzymatic glycation reaction of glucose, a) intracellular proteins such as hemoglobin A and lens crystallin, b) plasma proteins such as albumin, apoprotein, transferrin, haptoglobin, c) Examples thereof include enzymes such as cathepsins and pancreatic riboases, and d) glycated products of membrane proteins such as vascular endothelium and erythrocyte membrane proteins. Detection of glycated hemoglobin and glycated albumin is effective for detection evaluation and diagnosis of impaired glucose tolerance and diabetes, as well as cirrhosis of the liver. 2) For example, a) As a protein modified with a sugar chain related to a disease, a) AFP-L3 test performed using Lens culimaris agglutinin-A (LCA) to detect fucosylation of AFP is a highly accurate hepatocyte B) Detection of MUC1 that reacts with Wisteria floribunda agglutinin (WFA) is effective in screening for cancer derived from the biliary system, and c) Detection of M2BP that reacts with WFA. It is an effective test to evaluate the progression of fibrosis that progresses due to viral hepatitis. D) Aspergillus Oryzae I-fucose specific lectin (AOL) and Ricinus Communis Agglutinin (RCA-I) in α-1 acid glycoprotein Fibrosis occurring in the liver can be evaluated from the binding rate, and e) detection of sialylated sugar chain MUC1 in blood is an index for detecting interstitial pneumonia.
In any of the biomarker tests described above, each post-translational modification that occurs in a specific protein is quantitatively detected and measured, and then the ratio of the post-translational modification that is characteristic of the disease is calculated. The degree is evaluated. The reason for calculation based on the measurement results of 2 to 3 items is that a plurality of modified isomer proteins cannot be measured simultaneously in one reaction system. In view of such a situation, in the following example 1), the glycated serum albumin was directly detected, and in 2) the sugar chain modification in the glycoprotein was simultaneously detected.
1) Detection of glycated albumin:
According to the protocol of Dynabeads Protein G, 200 μl of anti-human albumin antibody adjusted to 5 μg / ml with PBS was reacted with 1.5 mg of Dynabeads Protein G for 10 minutes to prepare Dynabeads to which the antibody was bound.
Next, after reacting with 500 μl of a blood sample solution containing glycated albumin at room temperature for 2 hours, the antigen was recovered with Dynabeads to which the antibody was bound. After thoroughly washing, the mixture was reacted with 100 μg of SWCNT-antibody human albumin antibody at room temperature for 2 hours and then thoroughly washed with PBS. SWCNT-antibody bound to Dynabeads Quantitatively measures the near-infrared emission of SWCNT derived from human albumin antibody to quantify the amount of albumin in serum. The ratio of glycated albumin to albumin is calculated using light absorption characteristic of albumin and glycated albumin captured at 1000 nm to 1400 nm.
2) Detection of proteins modified with sugar chains associated with disease:
According to the protocol of Dynabeads ProteinG, 200 μl of anti-human α-1 acid glycoprotein antibody (or anti-M2BP antibody or anti-MUC1 antibody) adjusted to 5 μg / ml with PBS was reacted with 1.5 mg of Dynabeads ProteinG for 10 minutes. Adjusted the combined Dynabeads. Next, after reacting with 500 μl of the blood sample solution at room temperature for 2 hours, the antigen was recovered with Dynabeads to which the antibody was bound. The collected Dynabeads were thoroughly washed, and then biotinylated WFA or MAL-II, AOL, RCA-I was reacted at room temperature for 20 minutes. After thoroughly washing with PBS, SWCNT-added streptavidin was reacted, and the binding state of the lectin was measured by luminescence. In such a method, since it is not necessary to perform a special pretreatment on the sample, it is possible to detect the reactivity of a plurality of lectins in one biological sample.
For example, in order to simultaneously detect three different sugar chain-modified isomers found in α-1 acid glycoprotein, first, it is collected from a sample such as blood with Dynabeads to which an anti-human α-1 acid glycoprotein antibody is bound, By reacting SWCNT-added streptavidin with different excitation wavelengths and biotinylated AOL, biotinylated MAL-II, and biotinylated RCA-I at the same time, the three modified isomers can be reacted at the same time. It can be quantitatively evaluated.
In addition, by using SWCNT separated into three types having different emission wavelengths, and using three types of SWCNT-antibodies obtained by adding antibodies specific to the three types of antigens to be detected, It is possible to determine the abundance in the specimen for various types of proteins. Anti-human α-1 acid glycoprotein antibody, anti-M2BP antibody, and anti-MUC1 antibody-bound Dynabeads are used to recover the antigens against them, and the detection antibodies for detecting the recovered antigens are detected by SWCNTs with different excitation wavelengths. By labeling and using, three antigen amounts can be quantitatively evaluated simultaneously.
FIG. 7 is a schematic cross-sectional view of a whole blood spectroscopic fluorescence apparatus equipped with a small near infrared spectrometer. When the whole blood sample to which the SWCNT infrared fluorescent label is added is intermittently irradiated with near infrared light by a light emitting diode, the SWCNT emits fluorescence in synchronization with the light emitting diode light in the infrared wavelength band. The fluorescent light is condensed while removing the wavelength component of the illumination light with a low-pass filter and spectrally splitting into the near-infrared light detection array with the concave grating. In the figure, three systems of spectroscopic measurement systems having different spectral wavelength bands are arranged, and fluorescence and absorption in three wavelength bands are measured simultaneously in the same sample.

Current detection of biomarkers is limited by the wavelength of light used for signal detection, and is usually used in clinical tests.i) Plasma obtained by removing blood cell components (red blood cells, white blood cells, and platelets) from whole blood collected, or ii ) Serum obtained by coagulating blood to remove blood cell components and fibrin is measured as a sample. Although the method of removing by filter filtration is also taken in, these components cannot be removed by any operation, and hemoglobin is mixed by erythrocyte hemolysis caused by the operation. The contamination of these bio-derived substances obstructs and absorbs the luminescence (fluorescence) used in the disease biomarker detection evaluation and lowers the disease detection sensitivity. At the same time, blood cell components emit light non-specifically, which causes noise when detecting probe luminescence. Such interference and absorption of probe luminescence by autologous blood cell components and self-luminous noise interfere with high-sensitivity detection of disease biomarkers for the purpose of early diagnosis, and are very serious obstacles to their practical application.
FIG. 8 compares the spectrum measured by the apparatus shown in FIG. 7 with the spectrum in the conventional method. In the prior art, since the emission wavelength of the fluorescent label is at most 800 nm, the self-emission spectrum of hemoglobin will cover the fluorescence due to the label as background light, so first of all, using a large pretreatment device such as a centrifuge Need to remove red blood cells. In addition, since the emission wavelength range of the fluorescent label is narrow, normally only one wavelength is used for one detection, and therefore it is necessary to prepare the same number of specimens in order to detect a plurality of antigens.
On the other hand, the fluorescence detection device according to the present invention can detect fluorescence at a wavelength longer than the emission band of hemoglobin with high sensitivity, so that the sample can be introduced into the analysis device as whole blood, which is complicated. There is a feature that pre-treatment is unnecessary, that there is no influence of hemolysis or the like at the time of centrifugation or filtering, and as a result, sensitivity is improved by several tens of times.
In addition to liver function and cancer markers, by measuring light absorption up to a wavelength of 2 μm, blood sugar levels essential for monitoring lifestyle-related diseases can be measured at low cost.
Furthermore, spectroscopic measurement can be performed in a plurality of visible and infrared emission bands, and several types of antigens can be collected and detected in one reaction pot. As a result, there are features such as a small amount of specimen required and a low running cost.
FIG. 9 shows an example in which three sets of light emitting diodes, filters, and single light receiving elements are arranged instead of the small spectroscope. Since this device is simple and inexpensive, it can be used for clinical examinations in clinics and private hospitals.
FIG. 10 shows a schematic diagram of an apparatus for automatically processing the sample processing such as sample dispensing, antibody addition, and magnetic bead washing described in the present invention, and detecting weak fluorescence at near infrared wavelengths. Background light caused by hemoglobin can be removed by a relatively simple process.

Claims (15)

  An immunoassay method characterized by utilizing unique optical properties (light absorption, emission, Raman scattering light) of nanocarbon (single-walled carbon nanotube, multi-walled carbon nanotube, carbon nanohorn, nanographene, and other nanocarbons) Or clinical laboratory methods.   The immunoassay method or clinical test method according to claim 1, wherein single-walled carbon nanotubes (SWCNT) or nanographene that emit light at a wavelength of 700-2300 nm by photoexcitation at wavelengths of 450-1350 nm are used as a luminescent label.   Luminescent labeling substance containing nanocarbon (single-walled carbon nanotube, multi-walled carbon nanotube, carbon nanohorn, nanographene, other nanocarbon).   The luminescent labeling substance according to claim 3, wherein the nanocarbon is a single-walled carbon nanotube (SWCNT) or nanographene that emits light at a wavelength of 700-2300 nm by photoexcitation with a wavelength of 450-1350 nm.   The luminescence according to claim 3 or 4, characterized in that nanocarbon is bound to an antibody recognizing a specific antigen, and the antigen-antibody reaction is detected by measurement of nanocarbon fluorescence, Raman light, and light absorption. Labeling substance.   It consists of streptavidin and streptavidin-related molecules bound to nanocarbon, and detects the reaction product of biotinylated antibody and streptavidin bound to antigen by measuring fluorescence, Raman light, and light absorption of nanocarbon. The luminescent labeling substance according to claim 3 or 4.   In order to detect an antibody bound to an antigen by binding nanocarbon to an immunoglobulin-binding protein such as Protein G or Protein A, the binding reaction between the antibody and the antibody-binding protein is determined by the nanocarbon fluorescence, Raman light, and light absorption. The luminescent labeling substance according to claim 3 or 4, which is detected by measurement.   When a specific antigen becomes normal or pathological, it is a variety of antigens (including cancer-related antigens such as proteins and sugar chains, and disease biomarkers) that are present in blood and body cavity fluids. The luminescent labeling substance according to claim 5, characterized in that it is characterized in that   For samples enriched using antibodies against various antigens bound to carriers such as magnetic beads, Sepharose, Sephadex, etc., the antigen detection is performed by nano-carbon near-infrared emission measurement after binding of the nanocarbon labeled antibody. The immunoassay method or clinical test method according to claim 1 or 2.   The antigen according to claim 1 or 2, wherein the antigen as a disease marker is labeled with a nanocarbon-labeled antibody and collected by electrophoresis, and then detected by near-infrared emission measurement of the nanocarbon. Immunoassay method or clinical test method.   The luminescent clinical test method according to claims 1 to 10 in a single specimen, or a combination thereof with another luminescent clinical test method, wherein a plurality of antigens are detected in different emission wavelength bands, Immunoassay method or clinical test method.   12. The immunoassay method or clinical test method according to claim 11, wherein the emission intensity ratio from a plurality of antigens in different emission wavelength bands is used in a single specimen.   The ultraviolet ray, visible light, near infrared light, and infrared light emission measurement according to claim 11 or 12, wherein a single or a plurality of excitation lights are modulated and a near infrared light emission is detected in synchronization therewith.   An immunoassay characterized by using a SWCNT-labeled antibody as a secondary antibody in place of peroxidase or alkaline phosphatase used as a secondary antibody in an enzyme immunoassay (ELISA).   A measurement method for measuring a plurality of antibody reactions in the same specimen by using a fluorescent label at another different emission wavelength in addition to the emission wavelength of SWCNT according to claim 2.