CN113820501A - Sandwich immunoassay kit - Google Patents

Sandwich immunoassay kit Download PDF

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CN113820501A
CN113820501A CN202011524278.0A CN202011524278A CN113820501A CN 113820501 A CN113820501 A CN 113820501A CN 202011524278 A CN202011524278 A CN 202011524278A CN 113820501 A CN113820501 A CN 113820501A
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solution
gold
nitrogen
drying
concentration
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熊良钟
熊清爵
李庆海
张茂峰
熊孟智
王梓光
张超卫
颜亚伟
曹珍利
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Bozhou New Health Technology Co ltd
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Abstract

The invention provides an application of a surface-enhanced Raman detection kit in myoglobin detection, wherein a top layer reagent, a bottom layer reagent, a BSA solution and an EDTA anticoagulant are adopted in the kit, and the top layer reagent in the kit adopts 3-mercapto-1, 2, 4-triazole molecules as Raman detection molecules, so that the kit has a stronger function of strengthening Raman signals compared with the commonly used P-ATP molecules in the prior art; the surface-enhanced Raman detection kit is applied to human body endomyoglobin detection for the first time, can make up the defects in the prior detection technology, greatly improves the detection limit and sensitivity, and plays a key role in preventing sepsis.

Description

Sandwich immunoassay kit
Technical Field
The invention relates to the field of biochemical detection, in particular to a sandwich immunoassay kit.
Background
Neutrophil gelatinase-associated lipocalin (NGAL), also known as human lipocalin 2(lipocalin2, Ln2) or siderocalin (siderocalin), is a new member of the human lipocalin family, and a small molecular weight secretory protein, which is originally found in activated neutrophils, can participate in the transport of iron-containing substances, and the expression level in vivo is an effective index for the treatment and disease monitoring of different kidney diseases, the promotion of cell proliferation after kidney injury, and the inhibition of apoptosis. NGAL induces regeneration of renal tubular epithelial cells upon renal injury. Modern studies have shown that NGAL is one of the most effective biological markers for diagnosing acute kidney injury and also one of the effective markers for early diabetic nephropathy.
The beta 2-microglobulin is a small molecular globulin produced by lymphocyte, platelet and polymorphonuclear leukocyte, has molecular mass of 11800, and is a single-chain polypeptide consisting of 99 amino acids. It is the beta chain (light chain) part of cell surface Human Leukocyte Antigen (HLA), and contains a pair of disulfide bonds in the molecule and no sugar; similar to the structure of immunoglobulin constant region. Widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum. The synthesis rate and the release amount of the normal human beta 2-microglobulin from cell membranes are quite constant, the beta 2-microglobulin can be freely filtered from glomeruli, 99.9 percent of beta 2-microglobulin is absorbed by a proximal tubule and is decomposed and destroyed in a renal tubular epithelial cell; thus, the excretion of beta 2-microglobulin is normally very slight.
Clinically, the rise of serum beta 2-microglobulin can reflect the condition whether glomerular filtration function is damaged or filtration load is increased; increased excretion of beta 2-microglobulin from the urine would indicate increased renal tubular damage or filtration load; in acute and chronic pyelonephritis, the kidney is damaged, so the urine beta 2-microglobulin is increased, and the beta 2-microglobulin is normal for patients with cystitis; the beta 2-microglobulin in blood and urine of a patient with kidney transplantation is obviously increased, which indicates that the organism has rejection reaction, and the beta 2-microglobulin in blood is still increased although the kidney clearance is increased due to the accelerated synthesis of the beta 2-microglobulin. The blood beta 2-microglobulin generally rises to a peak 2-3 days after transplantation and then gradually falls. After kidney transplantation, continuous measurement of blood and urine beta 2-microglobulin can be used as sensitive index of glomerular and renal tubule diseases. For example, kidney transplantation shows that the prognosis is good although there is oliguria, but the blood beta 2-microglobulin is reduced. The beta 2-microglobulin is increased before Cr is measured during the rejection, which is helpful for diagnosing the rejection reaction of the kidney in the subclinical stage. The urine beta 2-microglobulin determination is also helpful for identifying the upper and lower urinary tract infection, the upper urinary tract infection is easy to influence the reabsorption of the molecular protein by the renal tubules, the urine beta 2 microglobulin is increased, and the urine beta 2 microglobulin is not increased when the lower urinary tract is infected.
Acute Myocardial Infarction (AMI) is ischemic necrosis of the local myocardium caused by acute occlusion of coronary arteries and interruption of blood flow. The typical symptoms of the disease are precordial pain or oppression, the pain or oppression mainly refers to the back of the sternum, extends downwards to the left rib and the upper abdomen, and extends upwards to the left shoulder, back, even mouth and head, and part of the pain or oppression can be shown in the left upper limb. The pain is mostly 'pressure, pressure and heaviness' in the precordial region, and some patients have no obvious feeling on the chest, and only dull pain or discomfort is caused in other parts except the chest. The pain lasts for a long time, more than 30min, no obvious pain peak exists, and the symptoms cannot be quickly relieved by quiet rest or application of medicines such as nitroglycerin and the like.
Since 2002, the incidence rate of acute myocardial infarction generally rises, compared with the prior art, the incidence rate of acute myocardial infarction in rural areas is obviously enhanced, the incidence rate of acute myocardial infarction in rural areas in part of years even exceeds that in cities, 2016, the death rate of acute myocardial infarction in cities is 58.69/10 ten thousand, and the death rate in rural areas is 74.72/10 ten thousand. In addition, the incidence of acute myocardial infarction is on the rise year by year in people under 45 years old, and the incidence is on the fall year by year in people above 45 years old.
Acute myocardial infarction patients are characterized by sudden attack, precordial compression pain or suffocation feeling lasting for more than 30min, and patients often feel dying. Therefore, early diagnosis and early negative exclusion of acute myocardial infarction play an important role, and the prognosis of acute myocardial infarction is also an important link, which is important for judging the recovery, complications, relapse and death probability of patients after treatment.
Studies have shown that cardiac fatty acid binding protein (hFABP) is a novel small cytoplasmic protein abundant in the heart. It is highly heart specific (i.e., it is expressed primarily in heart tissue), but is also expressed at low concentrations in tissues other than the heart. After ischemic myocardial injury occurs, type fatty acid binding protein (hFABP) can be found in the blood as early as 1-3 hours after the onset of chest pain, peaks at 6-8 hours and returns to normal plasma levels within 24-30 hours.
Type fatty acid binding protein (hFABP) appears similar to that of myoglobin, and these two low molecular weight cytoplasmic proteins expressed in cardiac and skeletal tissues are substrates for mitochondrial oxidation and are released within 2 hours after onset of symptoms, with maximum concentrations occurring at 6 hours and baseline concentrations returning within 24 hours. However, their concentrations in the heart and muscle tissue differ. The concentration of midsize fatty acid binding protein (hFABP) in the heart is 2-10 times higher than in skeletal muscle, in contrast to 2 times lower myoglobin concentration in heart cells than in skeletal cells. Although myoglobin was recommended as an early marker of cardiac injury in several published guidelines, the fatty acid binding protein type (hFABP) is more cardiac specific and due to its biological properties it may be considered a more accurate test for the diagnosis of acute myocardial infarction.
Research shows that the cardiac troponin I is used as a gold standard for AMI diagnosis, and AMI judges infarct area and carries out risk stratification. Clinically significant, CTNI can be used as a definitive marker for the diagnosis of myocardial infarction, risk stratification and patient prognosis assessment, to aid in clinical setting, or to adjust treatment regimens in time. CTNI has become widely accepted by clinicians and testers as the "gold standard" for the diagnosis of myocardial injury, particularly acute myocardial infarction. Troponin, which is composed of I, T, C trisubunits, is released into the blood to be elevated after myocardial injury, and the elevated CTNI can be maintained in the blood for a long period of time (5-10 days), thus providing a long detection period. Detection of micro-myocardial damage: the content of troponin I in blood is obviously increased when the cardiac muscle is slightly injured, and the troponin I can be detected within 4-6 hours, so that the sensitivity is high. CTNI has myocardial specificity and sensitivity, and is an important marker for diagnosing myocardial infarction. CTNI is a good marker for diagnosing myocardial damage of hypothyroidism patients. And (3) observation of the effect of the medicine: CTNI has also been used to observe the pharmacological effects of certain drugs on the heart to see if they improve or exacerbate the phenomenon of myocardial ischemia. When muscle tissues other than the myocardium are damaged or diseased, the creatine kinase isoenzyme CK-MB may be elevated, and CTNI may not exceed the critical value. Due to their minimal content in normal serum, there was a significant increase in AMI, and the fold increase generally exceeded the change in CK-MB. Because of its low molecular weight, CTNI, free after onset, is rapidly released from the myocardial cytoplasm and increases in blood concentration, at a time comparable to or slightly earlier than CK-MB. Although CTNI has a short half-life, it has a long duration of degradation from myofibrils, and can maintain a long time rise in blood, so it has the advantages of both an earlier CK-MB rise and a long diagnosis time window of LD 1. Therefore, CTNI has gradually replaced the enzyme index.
Research shows that the creatine kinase isozyme is a marker for early diagnosis of AMI and risk stratification, and non-ST-segment elevation of MI is the most valuable, which is beneficial to early diagnosis and risk stratification of acute coronary syndrome; estimating myocardial ischemia injury area; monitoring myocardial damage caused by various reasons; evaluation of clinical treatment effect; differential diagnosis of various chest pain causes.
Creatine kinase is an important energy metabolism enzyme in cells, is widely distributed, and is most abundant in muscle cells, and two subunits form a dimer; creatine kinase isoenzyme (CK-MB) is mainly present in the outer plasma layer of cardiomyocytes and has been the most specific enzyme in the myocardial zymogram for clinical diagnosis of myocardial injury. Creatine Creatinase (CK) began to rise 6h after onset, reached peak 24h and returned to normal 3-4 h. CK has three isoenzymes, CK-BB, CK-MM and CK-MB, wherein the latter is peculiar to cardiac muscle, and CK-MB is increased during AMI, thus having important significance for diagnosing AMI. The relevant markers should be detected synchronously when detecting the myocardial damage markers. The release time of the three markers Myo, cTnI and CK-MB is different, and the three markers are rapidly determined simultaneously, so that the method is more convenient and faster than the independent determination, and the time synchronization is more favorable for analyzing and judging the result, thereby being capable of achieving rapid and accurate diagnosis.
Myoglobin (Myo) has functions of oxygen transportation and oxygen storage in muscles, has small molecules, and can directly enter blood circulation without lymph nodes, so that cardiac muscle is directly entered into blood circulation from cardiac muscle cells when being slightly damaged. Myoglobin is a protein having an enzyme binding function, mainly present in cardiac muscle and skeletal muscle, and is released into the blood upon damage of the skeletal muscle and cardiac muscle, Acute Myocardial Infarction (AMI), hypermotility, and muscle diseases. Therefore, the myoglobin (Myo) concentration can be used as an early diagnosis index of acute myocardial infarction and also can be used as an index of coronary artery recanalization condition of thrombolytic therapy, and the myoglobin concentration reaches the highest after recanalization for 30 minutes to 2 hours.
The N-terminal pro-brain natriuretic peptide (NT-proBNP) is a peptide segment without biological activity, which is mainly formed by degrading pro-brain natriuretic peptide secreted by ventricular myocytes, and meanwhile, the pro-brain natriuretic peptide with biological activity is also produced in an equimolar way, and the two have the same clinical significance. The european cardiology society recommends the diagnosis of heart failure using brain natriuretic peptides and N-terminal brain natriuretic peptide precursors in combination with X-rays, echocardiograms, imaging, clinical manifestations, etc., and uses them as exclusion tests for heart failure. However, as a biomarker of cardiac function damage, compared with brain natriuretic peptide, the N-terminal brain natriuretic peptide precursor has longer half-life and better stability and sensitivity, so the N-terminal brain natriuretic peptide precursor is the only objective biochemical index for acute heart failure diagnosis at present, is the best index for managing heart function change diseases, can be used for screening, diagnosing, monitoring disease conditions and prognosis evaluation of patients with potential heart failure risk factors, asymptomatic heart failure, symptomatic heart failure and late-stage heart failure diseases, and is the most important judgment index in heart function change diseases, namely the ideal clinical marker of heart failure.
Whole range C-reactive protein refers to some acute proteins that rise sharply in plasma when the body is infected or tissue damaged. The whole course C-reactive protein can activate complement and strengthen phagocytosis of phagocyte to play a role in opsonization, thereby eliminating pathogenic microorganisms invading the body and damaged, necrotic and apoptotic histiocytes, and playing an important role in protection in the natural immune process of the body.
Meanwhile, the whole course C-reactive protein is not only a non-specific inflammation marker, but also directly participates in cardiovascular diseases such as inflammation and atherosclerosis, and is the strongest powerful predictor and risk factor for cardiovascular diseases. The interaction of the global C-reactive protein with complement Clq and FcTR allows it to exhibit a number of biological activities, including host defense against infection, phagocytosis and regulation of inflammatory responses, etc. The combination with damaged cells, apoptotic cells and nuclear antigens makes them play an important role in autoimmune diseases.
Sepsis occurs at a high rate, with over 1800 million severe sepsis cases worldwide per year, and this figure also rises at a rate of 1.5% to 8.0% per year. Sepsis is extremely morbid and has a high mortality rate, with about 14,000 people dying from its complications every day worldwide. According to foreign epidemiological investigation, the mortality rate of sepsis exceeds that of myocardial infarction, and becomes a main cause of death of non-cardiac patients in intensive care units. In recent years, despite significant advances in anti-infective therapy and organ function support technologies, sepsis has still suffered from a mortality rate of up to 30% to 70%. Sepsis treatment costs high, medical resources are consumed greatly, the quality of life of human beings is seriously affected, and great threats are already caused to human health. Thus, in 2001 the european severe society, the american severe society, and the international sepsis forum for sepsis "rescue of sepsis battles" (SSC) were initiated, and in 2002 a number of organizations in europe and america initiated and signed the "barcelona declaration" together, and further established and continuously updated the sepsis treatment guideline, i.e., SSC guideline, based on evidence-based studies on sepsis to improve the treatment of sepsis and reduce the mortality of sepsis. The SSC guidelines were first established in 2003 and later revised again in 2008.
Sepsis, severe sepsis (severe septis) and septic shock (septic shock) can be classified according to the severity of sepsis. Severe sepsis, refers to sepsis with organ dysfunction, poor tissue perfusion, or hypotension. Septic shock, which refers to severe sepsis given sufficient fluid to resuscitate and still be accompanied by uncorrectable persistent hypotension, is also considered a particular type of severe sepsis. Sepsis can be caused by infection at any site, and is clinically common in pneumonia, peritonitis, cholangitis, urinary system infection, cellulitis, meningitis, abscess, and the like. The pathogenic microorganisms comprise bacteria, fungi, viruses, parasites and the like, but not all patients with sepsis have positive blood culture results of the pathogenic microorganisms causing infection, and only about 45 percent of patients with septic shock can obtain the positive blood culture results. Sepsis often occurs in patients with severe disease, such as severe burns, multiple wounds, post surgical procedures, and the like. Sepsis is also common in patients with chronic diseases such as diabetes, chronic obstructive bronchi, leukemia, aplastic anemia, and urinary tract stones.
The most effective method for treating and preventing sepsis is based on the pathogenesis of sepsis, but unfortunately, the pathogenesis of sepsis is not completely clarified at present, and in this case, aiming at the pathogeny, clinical prevention work is done in all aspects, and efforts to reduce the risk factor inducing infection have important effects on the treatment and prevention of sepsis.
Procalcitonin (PCT) is a protein whose levels in plasma are elevated when severe bacterial, fungal, parasitic infections and sepsis and multi-organ failure. PCT does not rise upon autoimmunity, allergy and viral infection. Localized limited bacterial infection, mild infection and chronic inflammation did not result in elevation. Bacterial endotoxins play a crucial role in the induction process. PCT reflects the activity of the systemic inflammatory response.
At present, the commonly used detection modes of neutrophil gelatinase-associated lipocalin (NGAL) are a colloidal gold method and a fluorescence immunoassay method, the sensitivity can reach pg level, and the neutrophil gelatinase-associated lipocalin (NGAL) is used as a preventive detection index, has higher requirements on the detection sensitivity, and can achieve the purposes of early discovery, early prevention and early treatment of acute kidney injury and diabetic nephropathy.
At present, beta 2-microglobulin detection modes include radioimmunoassay, enzyme-linked immunosorbent assay and immunoturbidimetry, but the methods cannot simultaneously have the functions of low detection limit, simple operation and dynamic monitoring.
Currently, sandwich enzyme-linked immunosorbent assay, immunochromatography, and the like are mainly used for the measurement of heart-type fatty acid binding protein (hFABP). The heart-type fatty acid binding protein is used as an important preventive index, is more important for detecting the high sensitivity of the heart-type fatty acid binding protein, can achieve the effect of early finding and early treating, radically reduces the incidence probability of acute myocardial infarction, or avoids the occurrence of diseases. However, the detection methods in the prior art cannot satisfy the detection of heart-type fatty acid binding protein with high sensitivity.
At present, the troponin is mainly determined by a double-antibody sandwich immunological method, and the detection method comprises chemiluminescence, electrochemiluminescence and the like. The cardiac troponin I is used as an important preventive index, is more important for detecting the high sensitivity of the cardiac troponin I, can achieve the effect of early finding and early treating, radically reduces the incidence probability, or avoids the occurrence of diseases. However, the detection methods in the prior art do not satisfy the detection of high sensitivity to cardiac troponin I.
Currently, the commonly used creatine kinase isoenzyme (CK-MB) assay methods are: latex agglutination test, fluorescence immunoassay and ELISA, but creatine kinase isoenzyme (CK-MB) is an important preventive index, and is more important for detecting the creatine kinase isoenzyme with high sensitivity, so that the effect of early detection and early treatment can be achieved, the morbidity probability can be fundamentally reduced, or the occurrence of diseases can be avoided. Therefore, the detection method in the prior art cannot satisfy the detection of creatine kinase isoenzyme (CK-MB) with high sensitivity.
Currently, the commonly used myoglobin (Myo) determination methods include RIA method, latex agglutination test, two-site immunoassay, immunoturbidimetry, fluorescence immunoassay, ELISA method and high pressure liquid chromatography, but myoglobin is used as an important preventive index, and is more important for the high-sensitivity detection of myoglobin, so that the effect of early detection and early treatment can be achieved, the incidence of diseases can be fundamentally reduced, or the occurrence of diseases can be avoided. Therefore, the detection method in the prior art cannot satisfy the detection of high sensitivity to myoglobin (Myo).
At present, the detection mode of the N-terminal pro-brain natriuretic peptide comprises a colloidal gold method and an immunochromatography method, the detection limit is pg level, and the concentration of the N-terminal pro-brain natriuretic peptide is increased along with the severity of heart failure, so that the method has great significance for detecting the N-terminal pro-brain natriuretic peptide with higher sensitivity, and can prevent the occurrence of the heart failure early.
At present, the detection methods commonly used for the whole course C reactive protein are an immunodiffusion method, a radioimmunoassay, a nephelometry method and an enzyme-labeled immunoassay method, the detection limit of the methods is mu g, although the whole course C reactive protein has a normal range of 800-.
At present, the common detection modes of Procalcitonin (PCT) are a radioimmunoassay, a double-antibody sandwich immunochemiluminometry, a colloidal gold colorimetry and a transmission immunoturbidimetry, wherein the double-antibody sandwich immunochemiluminometry is the most common method at present and is the most convenient to operate, but the detection limit is 0.1ng/mL, the more sensitive the detection is to the preventive detection index procalcitonin, the lower the detection limit can be reached, and the better the preventive and therapeutic effects are.
Disclosure of Invention
In order to solve the problems in the prior art, an aspect of the present invention provides a sandwich immunoassay kit, which is characterized in that the sandwich immunoassay kit comprises: 10ml of top layer reagent, 10ml of bottom layer reagent, 10ml of BSA solution and 10ml of anticoagulant LEDTA;
the top layer reagent comprises: the method comprises the following steps of (1) preparing a gold-core silver-shell nanorod substrate, a cysteamine modified molecule, a glutaraldehyde modified molecule and a 1 XPBS buffer solution;
the bottom layer reagent comprises: a gold core silver shell nanorod substrate, 3-mercapto-1, 2, 4-triazole Raman detection molecules and glutaraldehyde functional modification molecules;
the concentration of the BSA solution was 1%.
Further, the preparation method of the sandwich immunoassay kit comprises the following steps:
(1) preparation of Top layer reagent
(1.1) adding a Raman detection molecular ethanol solution into a gold-core silver-shell nanorod solution, gently shaking and centrifuging, and removing a supernatant to obtain a first mixture;
(1.2) dispersing the mixture I into deionized water, adding a functional modifier solution, gently shaking and centrifuging to obtain a mixture II;
(1.3) dispersing the mixture II into a neutrophil gelatinase-associated lipocalin detection antibody solution to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12 hours, then centrifuging, removing a supernatant, dispersing a precipitate into a 1 x PBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) performing amino functionalization treatment on the gold-core silver-shell nanorod solution by using a cysteamine solution, washing with purified water after the treatment, and drying with nitrogen;
(2.2) adding a functional modifier solution for functional treatment, flushing with purified water after treatment, and drying with nitrogen;
(2.3) adding the product obtained in step 2.1 to a neutrophil gelatinase-associated lipocalin capture antibody solution, incubating at 4 ℃ for 12 hours, then washing with purified water, and drying with nitrogen.
Further, in the step (1.1), the Raman detection molecule is 3-mercapto-1, 2, 4-triazole, the molar concentration of the Raman detection molecule ethanol solution is 5mmol/L, and the dosage is 2 muL;
the dosage of the gold-core silver-shell nanorod solution is 1 mL;
the time for the gentle shaking was 2h and the conditions for the centrifugation were 7000rpm, 10 min.
Further, the amount of the deionized water used in the step (1.2) is 1.0 mL;
the functionalized modifier solution is glutaraldehyde solution, the concentration is 25% wt, and the dosage is 2 mu L;
the time for the gentle shaking was 1.5h, and the centrifugation conditions were 6000rpm, 10 min.
Further, the antibody solution for detecting neutrophil gelatinase-associated lipocalin in step (1.3) is a polyclonal antibody solution against neutrophil gelatinase-associated lipocalin, the concentration is 9 μ g/mL in 1 × PBS buffer, and the dosage is 1.0 mL;
the centrifugation condition is 6000rpm for 10 min;
the amount of the 1 XPBS buffer was 1.0 mL.
Further, the concentration of the cysteamine solution in step (2.1) is 25mmol/L, and the dosage is 2. mu.L.
Further, the solution of the functional modifier in step (2.2) is glutaraldehyde solution with a concentration of 25% wt, and the amount is 2 μ L.
Further, the neutrophil gelatinase-associated lipocalin capture antibody solution in step (2.3) is a neutrophil gelatinase-associated lipocalin monoclonal antibody solution, the concentration is 20 μ g/mL in 1 × PBS buffer, and the dosage is 2 mL.
Another aspect of the present invention provides a method for detecting neutrophil gelatinase-associated lipocalin using a sandwich immunoassay kit, the method comprising:
(a) blocking non-specific binding
Firstly, immersing a bottom layer reagent into a BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized neutrophil gelatinase-associated lipocalin
Dripping 200 mu L of detection sample into the bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water, and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on a Leica microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
Further, the detection sample is one of whole blood, plasma or serum;
when the detection sample is whole blood, an EDTA anticoagulant needs to be added before detection for anticoagulation.
Compared with the prior art, the invention has the following advantages:
the invention provides a sandwich immunoassay kit, which adopts gold-core silver-shell nanoparticles to bond Raman detection molecules and detection antibodies to obtain a sandwich structure top layer solution, and adopts gold-core silver-shell nanoparticles to bond capture antibodies to obtain a sandwich structure substrate solution, and the method is easy to operate and low in cost, and the kit prepared by the method adopts 3-mercapto-1, 2, 4-triazole molecules as Raman detection molecules, and has a stronger function of strengthening Raman signals compared with P-ATP molecules commonly used in the prior art; the sandwich immunoassay kit is used for detecting the neutrophil gelatinase-associated lipocalin, is simple and convenient to operate and high in sensitivity, and can be used for rapidly detecting the target protein.
Drawings
FIG. 1 is a diagram showing the process of forming a sandwich immunoassay kit structure according to the present invention.
FIG. 2 is a Raman spectrum of neutrophil gelatinase-associated lipocalin according to the first embodiment of the present invention.
FIG. 3 is a Raman spectrum of different concentration levels of neutrophil gelatinase-associated lipocalin in a first embodiment of the invention.
FIG. 4 is a Raman spectrum of β 2-microglobulin according to a second embodiment of the present invention.
FIG. 5 is a Raman spectrum of β 2-microglobulin at various concentration levels according to a second embodiment of the present invention.
FIG. 6 shows a Raman spectrum of a cardiac fatty acid binding protein according to a third embodiment of the present invention.
FIG. 7 is a Raman spectrum of cardiac fatty acid binding protein at different concentration levels according to a third embodiment of the present invention.
FIG. 8 shows a Raman spectrum of cardiac troponin I in the fourth example of the present invention.
FIG. 9 shows Raman spectra of cardiac troponin I at different concentration levels according to the fourth embodiment of the present invention.
FIG. 10 is a Raman spectrum of creatine kinase isoenzyme in the fifth example of the present invention.
FIG. 11 shows Raman spectra of creatine kinase isozyme at different concentration levels in the fifth example of the present invention.
FIG. 12 shows a Raman spectrum of myoglobin in the sixth example of the present invention.
FIG. 13 shows Raman spectra of myoglobin at different concentration levels according to the sixth embodiment of the present invention.
FIG. 14 is a Raman spectrum of an N-terminal pro-brain natriuretic peptide according to a seventh embodiment of the present invention.
FIG. 15 is a Raman spectrum of N-terminal pro-brain natriuretic peptide at different concentration levels according to a seventh embodiment of the present invention.
FIG. 16 is a Raman spectrum of a whole course C-reactive protein according to an eighth example of the present invention.
FIG. 17 is a Raman spectrum of a whole course C-reactive protein at various concentration levels according to the eighth example of the present invention.
FIG. 18 is a Raman spectrum of procalcitonin in the ninth embodiment of the invention.
Fig. 19 is a raman spectrum of different concentration levels of procalcitonin in a ninth embodiment of the invention.
Detailed Description
The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Embodiments of the present invention will hereinafter be described with reference to the accompanying drawings, wherein like reference numerals denote like or similar parts, or like or similar steps.
The first embodiment.
As shown in fig. 1, the process of forming a sandwich immunoassay kit structure of the present invention is illustrated, and the present embodiment provides a method for preparing a sandwich immunoassay kit, which includes the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of neutrophil gelatinase-associated lipocalin sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of a neutrophil gelatinase-associated lipocalin polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing the precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a monoclonal antibody solution of neutrophil gelatinase-associated lipocalin at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried with nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the neutrophil gelatinase-associated lipocalin sandwich immunoassay kit prepared by the method, and 10mL of 1% BSA solution is provided in the kit.
FIG. 2 shows a Raman spectrum of neutrophil gelatinase-associated lipocalin in a first embodiment of the invention. Patient sera were tested using the neutrophil gelatinase-associated lipocalin sandwich immunoassay kit of the examples.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the neutrophil gelatinase-associated lipocalin sandwich immunoassay kit in the embodiment are taken to operate according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized neutrophil gelatinase-associated lipocalin
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 2, and the result shows that 1013cm is in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined primarily as the presence of neutrophil gelatinase-associated lipocalin that can be detected by the kit, and to further demonstrate its accuracy, and the detection limits of the kit, we performed the following assays.
FIG. 3 shows Raman spectra of neutrophil gelatinase-associated lipocalin at different concentration levels in the first embodiment of the invention. The neutrophil gelatinase-associated lipocalin sandwich immunoassay kit of the examples was used to detect different concentrations of neutrophil gelatinase-associated lipocalin.
First, 6 samples were prepared, and neutrophil gelatinase-associated lipocalin was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL, respectively, to obtain samples Nos. 1-6.
Then, 6 parts of the neutrophil gelatinase-associated lipocalin sandwich immunoassay kit in the examples are taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized neutrophil gelatinase-associated lipocalin
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The raman spectrum obtained is shown in fig. 2, and the result shows that the raman intensity shows a monotonous rising trend along with the increase of the concentration of the neutrophil gelatinase-associated lipocalin, so that the neutrophil gelatinase-associated lipocalin sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human neutrophil gelatinase-associated lipocalin, and the detection limit is in the fg level.
Example two.
This example utilizes a sandwich immunoassay kit to detect beta 2-microglobulin. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two, beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of beta 2-microglobulin (beta 2-MG) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a solution of the monoclonal antibody β 2-microglobulin (. beta.2-MG) at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried with nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit prepared by the method, and 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 4 shows a Raman spectrum of β 2-microglobulin of a second embodiment of the present invention. Patient sera were tested using the beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit of the example.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit in the embodiment is taken to operate according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized beta 2-microglobulin (beta 2-MG)
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 4, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the presence of beta 2-microglobulin (beta 2-MG), and in order to further prove the accuracy and detection limit of the kit, we proceed with the following determination.
FIG. 5 shows Raman spectra of β 2-microglobulin at different concentration levels in a second embodiment of the invention. Different concentrations of β 2-microglobulin (β 2-MG) were tested using the β 2-microglobulin (β 2-MG) sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and β 2-microglobulin (. beta.2MG) was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to obtain samples Nos. 1 to 6.
Then, 6 parts of the beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit in the example were taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized beta 2-microglobulin (beta 2-MG)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 4, and the result shows that the raman intensity shows a monotonous rising trend along with the increase of the concentration of the beta 2-microglobulin (beta 2-MG), so that the beta 2-microglobulin (beta 2-MG) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human beta 2-microglobulin (beta 2-MG), and the detection limit is in the fg level.
Example three.
This example utilizes a sandwich immunoassay kit to detect cardiac fatty acid binding proteins. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two-heart type fatty acid binding protein sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of anti-human heart fatty acid binding protein polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of a monoclonal antibody against human cardiac fatty acid binding protein, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the heart-type fatty acid binding protein sandwich immunoassay kit prepared by the method, and 10mL of 1% BSA solution is also provided in the kit.
FIG. 6 shows a Raman spectrum of cardiac fatty acid binding protein in the third example of the present invention. Patient sera were tested using the heart-type fatty acid binding protein sandwich immunoassay kit of the examples.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the heart-type fatty acid binding protein sandwich immunoassay kit in the embodiment is taken to operate according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human cardiac fatty acid binding proteins
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 6, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak value, but the absence of such a peak value in the blank sample, can be preliminarily determined that the kit can detect the existence of the human heart-type fatty acid binding protein, and in order to further prove the accuracy and detection limit of the kit, the following determination is carried out.
FIG. 7 shows Raman spectra of cardiac fatty acid binding proteins at different concentration levels according to the third embodiment of the present invention. Human heart-type fatty acid binding protein was detected at different concentrations using the heart-type fatty acid binding protein sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and human cardiac fatty acid binding proteins were added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the heart-type fatty acid binding protein sandwich immunoassay kit in the embodiment is prepared according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human cardiac fatty acid binding proteins
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 6, and the result shows that the raman intensity shows a monotonous rising trend along with the enhancement of the concentration of the human heart-type fatty acid binding protein antigen, so that the heart-type fatty acid binding protein sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human heart-type fatty acid binding protein, and the detection limit is in the fg level.
Example four.
This example utilizes a sandwich immunoassay kit to detect cardiac troponin I. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of cardiac troponin I sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of an anti-human cardiac troponin I polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of anti-human cardiac troponin I monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the cardiac troponin I sandwich immunoassay kit prepared by the method, and 10mL of 1% BSA solution is provided in the kit.
FIG. 8 shows a Raman spectrum of cardiac troponin I in the fourth example of the present invention. Patient sera were tested using the cardiac troponin I sandwich immunoassay kit of the examples.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the cardiac troponin I sandwich immunoassay kit in the embodiment are taken according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human cardiac troponin I
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 8, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, makes it possible to preliminarily confirm that the kit can detect the presence of human cardiac troponin I, and to further prove the accuracy thereof, and the detection limit of the kit, we proceed with the following assay.
As shown in fig. 9, raman spectra of cardiac troponin I at different concentration levels in the fourth embodiment of the present invention are shown. Human cardiac troponin I at different concentrations was detected using the cardiac troponin I sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and human cardiac troponin I was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to obtain samples Nos. 1 to 6.
Then, 6 parts of the cardiac troponin I sandwich immunoassay kit in the example is taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human cardiac troponin I
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 8, and the result shows that the raman intensity shows a monotonous rising trend along with the enhancement of the concentration of the human cardiac troponin I antigen, so that the cardiac troponin I sandwich immunoassay kit prepared in the embodiment of the present invention can detect the presence of the human cardiac troponin I, and the detection limit is at the fg level.
Example five.
This example utilizes a sandwich immunoassay kit to detect creatine kinase isoenzyme (CK-MB). The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two, creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of anti-human creatine kinase isoenzyme (CK-MB) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a monoclonal antibody solution against human creatine kinase isoenzyme (CK-MB) at a concentration of 20. mu.g/mL in 1 XPBS buffer, incubated at 4 ℃ for 12 hours, then washed with purified water and dried with nitrogen.
The top layer reagent 10mL and the bottom layer reagent 10mL of the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit prepared by the method, and 10mL of BSA solution with the concentration of 1% are provided in the kit.
FIG. 10 shows a Raman spectrum of creatine kinase isozyme in the fifth embodiment of the present invention. Patient sera were tested using the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit of the examples.
First, 10mL of patient serum was obtained as a test sample, then 10mL of calf serum was used as a blank sample, and then 2 parts of the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit in the example was used according to the following procedure:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized human creatine kinase isozyme (CK-MB)
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 10, and the results are shown in the TableClearly, 1013cm in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the presence of human creatine kinase isoenzyme (CK-MB), and in order to further prove the accuracy and detection limit of the kit, we performed the following assay.
Raman spectra of creatine kinase isozymes at different concentration levels in the fifth example of the invention are shown in figure 11. The creatine kinase isoenzyme (CK-MB) antigen was detected at different concentrations using the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit of the example.
First, 6 samples were prepared, and human creatine kinase isoenzyme (CK-MB) antigens were added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit in the example is prepared according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized human creatine kinase isozyme (CK-MB)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 10, and the result shows that the raman intensity shows a monotonous rising trend along with the enhancement of the antigen concentration of the human creatine kinase isoenzyme (CK-MB), so that the creatine kinase isoenzyme (CK-MB) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human creatine kinase isoenzyme (CK-MB) and the detection limit is in the fg level.
Example six.
This example utilizes a sandwich immunoassay kit to detect myoglobin. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of myoglobin sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of an anti-human myoglobin polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of anti-human myoglobin monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
The top layer reagent 10mL and the bottom layer reagent 10mL of the myoglobin sandwich immunoassay kit prepared by the method are provided, and in addition, 10mL of BSA solution with the concentration of 1% is also provided in the kit.
FIG. 12 shows a Raman spectrum of myoglobin in the sixth embodiment of the present invention. Patient sera were tested using the myoglobin sandwich immunoassay kit of the example.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the myoglobin sandwich immunoassay kit in the embodiment are taken according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human myoglobin
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 12, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, can be determined preliminarily that the kit can detect the presence of human myoglobin, and in order to further prove the accuracy thereof, and the detection limit of the kit, we performed the following assay.
FIG. 13 shows Raman spectra of myoglobin at different concentration levels in the sixth embodiment of the invention. The myoglobin sandwich immunoassay kit of the example was used to detect different concentrations of human myoglobin antigen.
First, 6 samples were prepared, and human myoglobin antigens at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL were added to 6 calf sera, respectively, to obtain samples Nos. 1 to 6.
Then, 6 parts of myoglobin sandwich immunoassay kit in the embodiment is taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human myoglobin
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 12, and the result shows that the raman intensity is in a monotonous rising trend along with the increase of the concentration of the human myoglobin antigen, so that the myoglobin sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human myoglobin, and the detection limit is in the fg level.
Example seven.
This example utilizes a sandwich immunoassay kit to detect the N-terminal pro-brain natriuretic peptide. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two-terminal and N-terminal brain natriuretic peptide precursor sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of N-terminal brain natriuretic peptide precursor polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing supernatant, dispersing the precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer N-terminal pro-brain natriuretic antibody solution, incubated at 4 ℃ for 12 hours, then rinsed with purified water and dried under nitrogen.
The top layer reagent 10mL and the bottom layer reagent 10mL of the N-terminal brain natriuretic peptide precursor sandwich immunoassay kit prepared by the method are provided, and 10mL of BSA solution with the concentration of 1% is provided in the kit.
FIG. 14 shows a Raman spectrum of an N-terminal pro-brain natriuretic peptide according to a seventh embodiment of the present invention. Patient sera were tested using the N-terminal brain natriuretic peptide precursor sandwich immunoassay kit of the examples.
Firstly, 10mL of patient serum is taken as a test sample, then 10mL of calf serum is taken as a blank sample, and then 2 parts of the N-terminal brain natriuretic peptide precursor sandwich immunoassay kit in the embodiment are taken according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of N-terminal brain natriuretic peptide precursors
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 14, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The presence of a distinct peak, but the absence of such a peak in the blank sample, makes it possible to establish preliminarily that the kit can detect the presence of the N-terminal pro-brain natriuretic peptide, and to further demonstrate its accuracy, and the detection limit of the kit, we proceed with the following assay.
Raman spectra of N-terminal pro-brain natriuretic peptide at different concentration levels according to the seventh embodiment of the present invention are shown in FIG. 15. Different concentrations of N-terminal pro-brain natriuretic peptide were detected using the N-terminal pro-brain natriuretic peptide sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and N-terminal pro-brain natriuretic peptide was added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL, respectively, to obtain samples Nos. 1 to 6.
Then, 6 parts of the N-terminal brain natriuretic peptide precursor sandwich immunoassay kit in the embodiment are taken and operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of N-terminal brain natriuretic peptide precursors
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 14, and the result shows that the raman intensity is in a monotonous rising trend along with the increase of the concentration of the N-terminal pro-brain natriuretic peptide, so that the N-terminal pro-brain natriuretic peptide sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human N-terminal pro-brain natriuretic peptide, and the detection limit is in the fg level.
Example eight.
This example utilizes a sandwich immunoassay kit to detect whole course C reactive protein. The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of two-course C-reactive protein sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture II into 1.0mL of the whole course C-reactive protein polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of the Whole course C-reactive protein monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of top layer reagent and 10mL of bottom layer reagent of the whole C-reactive protein sandwich immunoassay kit are prepared by the method, and in addition, 10mL of BSA solution with the concentration of 1% is also provided in the kit.
FIG. 16 shows a Raman spectrum of a whole course C-reactive protein in the eighth example of the present invention. Patient sera were tested using the whole course C-reactive protein sandwich immunoassay kit of the examples.
Firstly, 10mL of patient serum is obtained as a test sample, then 10mL of calf serum is used as a blank sample, and then 2 parts of the whole course C-reactive protein sandwich immunoassay kit in the embodiment are taken to operate according to the following steps:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of Whole course C reactive protein
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 16, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak value, but the absence of such a peak value in the blank sample, can be preliminarily determined that the kit can detect the existence of the C-reactive protein in the whole course, and in order to further prove the accuracy and the detection limit of the kit, the following determination is carried out.
FIG. 17 shows Raman spectra of whole range C-reactive protein at different concentration levels in the eighth example of the present invention. The whole course C-reactive protein sandwich immunoassay kit in the embodiment is used for detecting the whole course C-reactive protein with different concentrations.
First, 6 samples were prepared, and Whole range C-reactive proteins were added to 6 calf sera at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL and 0fg/mL, respectively, to give samples Nos. 1-6.
Then, 6 parts of the whole course C-reactive protein sandwich immunoassay kit in the embodiment is operated according to the following steps:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of Whole course C reactive protein
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in figure 16, and the result shows that the Raman intensity is in a monotonous rising trend along with the enhancement of the concentration of the whole course C-reactive protein, so that the whole course C-reactive protein sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the whole course C-reactive protein of a human, and the detection limit is in the fg level.
Example nine.
This example utilizes a sandwich immunoassay kit for the detection of Procalcitonin (PCT). The embodiment provides a preparation method of a sandwich immunoassay kit, which comprises the following steps:
preparation of gold-core silver-shell nano rod
1. Preparation of gold nanorods
1) Diluting 0.1mL of HAuCl4 solution with the concentration of 25mM to 5mL by deionized water in a 20mL glass bottle, and adding 5mL of 0.2M CTAB solution into the diluted solution to obtain a solution I;
2) quickly injecting 0.6mL0.01M NaBH4 solution into the solution I, preparing NaBH4 solution in situ, magnetically stirring the mixed solution at the speed of 1200rpm for 2min, and standing the obtained seed solution at 30 ℃ for 30min for later use;
3) dissolving 7.0g of CTAB and 1.234g of sodium oleate in 250mL of 50 ℃ water, naturally cooling to 30 ℃, adding 18mL of 4.0mM silver nitrate solution, and keeping the temperature for 1min to obtain a solution II;
4) injecting 250ml of 1.0mM HAuCl4 solution into the second solution while magnetically stirring, stirring at 700rpm for 90min, changing the second magnetic stirring speed to 400rpm, adding 2.1ml of 37 wt% HCl solution while stirring, stirring for 15min, finally adding 1.25ml of 0.064M ascorbic acid solution, stirring for the third time at 1200rpm, and stirring for 30s to obtain a growth solution;
5) injecting 0.4mL of seed solution into the growth solution, stirring at 1500rpm for 30s, standing the mixed solution at 30 ℃ for 10h, centrifuging the standing mixed solution at 8000r/min for 10min, collecting precipitate, and dispersing the precipitate in 80mM CTAC solution;
6) repeating the step 5) for three times, storing the obtained precipitate in a CTAC solution to obtain a gold nanorod solution
2. Preparation of gold-core silver-shell nanorod
Diluting 0.5mL of gold nanorod solution to 4mL with water, adding 2.5mL of 10mM silver nitrate solution into the diluent, carrying out ultrasonic treatment for 2min at the frequency of 1000Hz, then adding 2.5mL of 0.1M ascorbic acid solution, preserving in a water bath at 65 ℃ for 4h, centrifuging at 8000r/min for 10min, collecting precipitate, and dispersing in 1mL of deionized water to obtain the gold-core-silver-shell nanorod suspension.
Preparation of Procalcitonin (PCT) sandwich immunoassay kit
(1) Preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of anti-human Procalcitonin (PCT) polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of anti-human Procalcitonin (PCT) monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
10mL of a top layer reagent and 10mL of a bottom layer reagent of the Procalcitonin (PCT) sandwich immunoassay kit are prepared by the method, and 10mL of 1% BSA solution is also provided in the kit.
FIG. 18 shows a Raman spectrum of procalcitonin in the ninth embodiment of the invention. Patient sera were tested using the Procalcitonin (PCT) sandwich immunoassay kit of the examples.
First, 10mL of patient serum was taken as a test sample, then 10mL of calf serum was taken as a blank sample, and then 2 parts of the Procalcitonin (PCT) sandwich immunoassay kit in the example were taken according to the following procedure:
a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized human Procalcitonin (PCT)
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained Raman spectrum is shown in FIG. 18, and the result shows that 1013cm is included in the Raman spectrum curve of the test sample-1The existence of a distinct peak, but the absence of such a peak in the blank sample, makes it possible to determine preliminarily that the kit can detect the presence of human Procalcitonin (PCT), and to further prove the accuracy, and the detection limit of the kit, we proceed with the following assay.
As shown in fig. 19, raman spectra of procalcitonin at different concentration levels in the ninth embodiment of the invention. Various concentrations of human Procalcitonin (PCT) antigen were detected using the Procalcitonin (PCT) sandwich immunoassay kit of the examples.
First, 6 samples were prepared, and human Procalcitonin (PCT) antigens at concentrations of 1ng/mL, 10pg/mL, 100fg/mL, 1fg/mL, 0.1fg/mL, and 0fg/mL were added to 6 calf sera, respectively, to obtain samples Nos. 1-6.
Then, 6 parts of the Procalcitonin (PCT) sandwich immunoassay kit of the example were prepared according to the following procedure:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilized human Procalcitonin (PCT)
Respectively dropwise adding 200 mu L of No. 1-6 sample into the 6 parts of bottom layer reagent treated in the step (a), culturing in a greenhouse for 1h, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
And (b) after drying by using nitrogen, adding a top layer reagent, placing in a greenhouse for 20min, washing by using purified water after finishing cultivation, and drying by using nitrogen to obtain the sandwich structure.
(d) Spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 6 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
The obtained raman spectrum is shown in fig. 18, and the result shows that the raman intensity shows a monotonous rising trend along with the enhancement of the concentration of the human Procalcitonin (PCT) antigen, so that the Procalcitonin (PCT) sandwich immunoassay kit prepared in the embodiment of the invention can detect the existence of the human Procalcitonin (PCT), and the detection limit is in the fg level. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (3)

1. A sandwich immunoassay kit, comprising: 10ml of top layer reagent, 10ml of bottom layer reagent, 10ml of BSA solution and 10ml of anticoagulant LEDTA;
the top layer reagent comprises: the method comprises the following steps of (1) preparing a gold-core silver-shell nanorod substrate, a cysteamine modified molecule, a glutaraldehyde modified molecule and a 1 XPBS buffer solution;
the bottom layer reagent comprises: a gold core silver shell nanorod substrate, 3-mercapto-1, 2, 4-triazole Raman detection molecules and glutaraldehyde functional modification molecules;
the concentration of the BSA solution was 1%.
2. The sandwich immunoassay kit of claim 1, wherein the sandwich immunoassay kit is prepared by a method comprising:
(1) preparation of Top layer reagent
(1.1) adding 2 mu L of 3-mercapto-1, 2, 4-triazole ethanol solution with the concentration of 5mmol/L into the gold-core silver-shell nanorod solution, gently shaking for 2h, centrifuging at 7000rpm for 10min, and removing the supernatant to obtain a first mixture;
(1.2) dispersing the first mixture into deionized water, adding 2 mu L of glutaraldehyde solution with the concentration of 25% wt, gently shaking for 1.5h, and centrifuging at 6000rpm for 10min to obtain a second mixture;
(1.3) dispersing the mixture into 1.0mL of an anti-human myoglobin polyclonal antibody solution with the concentration of 9.0 mu g/mL in 1 XPBS buffer to obtain a mixed solution, storing the mixed solution at 4 ℃ for 12h, then centrifuging at 6000rpm for 10min, removing a supernatant, dispersing a precipitate into 1mL of 1 XPBS buffer solution to obtain a top layer reagent, and storing at 4 ℃;
(2) preparation of the underlying agent
(2.1) adding 2 mu L of cysteamine solution with the concentration of 25mmol/L into 1.0mL of gold-core silver-shell nanorod solution, carrying out amino functionalization treatment on the gold-core silver-shell nanorod solution, washing the treated gold-core silver-shell nanorod solution with purified water, and drying the treated gold-core silver-shell nanorod solution with nitrogen;
(2.2) adding 2 mu L of glutaraldehyde solution with the concentration of 25mmol/L to perform functionalization treatment, washing with purified water after treatment, and drying with nitrogen;
(2.3) the product obtained in step 2.2 was added to 2mL of a 20. mu.g/mL in 1 XPBS buffer solution of anti-human myoglobin monoclonal antibody, incubated at 4 ℃ for 12 hours, then washed with purified water and dried under nitrogen.
3. A method for detecting a myoglobin antigen using the sandwich immunoassay kit of claim 1 or 2, said method comprising:
(a) blocking non-specific binding
Immersing the bottom layer reagent into BSA solution for 1h, blocking non-specific binding active sites, then flushing with purified water, and drying with nitrogen;
(b) immobilization of human myoglobin
Respectively dripping 200 mu L of test sample and blank sample into 2 parts of bottom layer reagent treated in the step (a), culturing for 1h in a greenhouse, then flushing with purified water and drying with nitrogen;
(c) forming a sandwich structure
After drying by nitrogen in the step (b), adding a top layer reagent, placing in a greenhouse for 20min, washing by purified water after finishing cultivation, and drying by nitrogen to obtain a sandwich structure;
(d) spectrum collection
Taking 785nm laser as an excitation light source, and performing Raman spectrum collection on the 2 samples on a come card microscope by using a 20-time objective lens, wherein the spectrum is exposed for 10s in the range of 800-1800cm < -1 >.
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