TW200418987A - Coating of molecularly imprinted membranes on chips to recognize biomolecules - Google Patents

Abstract

Based on the direct formation of molecularly imprinted membrane on gold electrode, we have developed an immunosensor for the detection of biomolecules. A new cross-linking monomer, (N-Acr-L-Cys-NHBn)2 was employed to attach the surface of the chip and to copolymerize with other monomers. Using quartz crystal microbalance detection, peptides and virus proteins can be monitored by their interaction with plastic antibodies specific for the target peptides. The selectivity of molecularly imprinted polymer and the sensitivity of such artificial biosensors have collaborated to differentiate traces of peptides and proteins to the ng/ml scale. Molecularly imprinted membranes also demonstrate the high ability to reuse.

Description

200418987

Background of the invention:

To detect the substance in the solution, you can use the combination of instruments and technical methods to find the solution. For example, enzyme linked immUn0S0rbent assay (ElisA) or other methods that can be combined with antibodies. However, 'sometimes antibodies are not suitable for detecting substances. Furthermore, antibodies cannot be induced using south toxic substances or low molecular weight chemicals. In recent years, due to the dengue fever epidemic virus, acquired immunodeficiency virus (A 丨 DS), enterovirus, etc., there has been a significant expansion trend. Although the current prevention and treatment can extract body fluids to isolate the virus, polymerase chain reaction (PCR), or detect antibodies, etc., Both need to select the appropriate antigens and antibodies a few days after the onset to detect it, and in terms of timeliness and demand, the effect of preventing and preventing epidemics cannot be achieved. Therefore, there is an urgent need to have a new biosensor method, which can obtain the detection results within minutes, and has specific interaction and affinity, so that the sensor can exert high sensitivity 'to replace the traditional time-consuming and expensive detection , And can effectively prevent the spread of the epidemic, early treatment.

The biosensor uses im JJJ 〇bi 1 ized biomolecules in combination with a transducer to detect a certain free test molecule. Because the test molecule and the immobilized biomolecule can generate The specific affinity reaction, when these two molecules interact, the transducer can respond to the current (Huang Jincheng, Food Industry Monthly, 2 9 (8), 6 ~ 8, 1997). Quartz Crystal Microbalance (QCM) is a sensing device. After a biomolecule reacts with a specific molecule to be measured, it will change the oscillation frequency of the piezoelectric crystal on the QCM, so it can be measured. The mass of the test molecule

200418987 V. Description of the invention (2) Concentration has been applied to the analysis of different substances, because the substance on the side can be adsorbed on the surface of the piezoelectric quartz crystal, and the mass of the substance can be accurately changed by the change of the oscillator frequency Changes are measured. Konash and Nomura first applied QCM to microgravimetric analysis in liquids, so they started QCM as a microsensor in liquids (Konash PL el; al, Anal. Chem. 66:34 1 to 344, 1 994) ° Nomura research The group applied QCM to solve electrochemical problems in the liquid phase, which made solid-liquid interface analysis technology a powerful tool, and then had a significant impact on the research and development of chemical and biological sensors (Nomura T ·, Anal · Chim · Acta · 131: 97 ~ 102,

I 9 8 1). Since then, studies using Q C Μ as the main body as the biochemical analysis immunoprobe can enhance its frequency change through competitive immune response and latex agglutination (Nakamra C. et al, (2 002) Anal · Chim ·

Acta 469, 183 ~ 188). Due to its high sensitivity, simple operation, easy signal interpretation, and real-time measurement, QCM can be used to detect the kinetics of molecular reactions, oligonucleosides and protein bonds that are bound to the stationary phase by pept i de Receptor to stationary phase (recep 10 rs), medical detection (Su CC et al, (2003) Anal. Chim. Acta. 479, II 7-1 2 3), detection of microbial pathogens and other molecular identification elements on.

What many chemists are interested in is the development of selective non-natural materials. 'The general idea is based on molecular displacement or interaction-derived identification technology.' One of the methods of analysis is molecular printing. The method of manufacturing a polymer printed film can show selectivity and the ability to distinguish molecularly. The characteristics of these printed substrates (ma tri X) tend to be simple and cheap to make, and are not easy to change in nature.

200418987 V. Description of the invention (3) Identification of the molecule to be tested is based on the rational design or obtained from biological sources. The printing principle is to polymerize an organic (high-chain) or inorganic polymer in the presence of a template molecule to convert the template After the molecule is removed] the remaining polymer still retains complementary binding sites (Mndi called sites) to the template molecule, which has become a kind of benefit that can identify the existence of small molecules and large molecules. Molecules including amino acids, nucleic acids, sugars, lipids, tritium, proteins, antibodies, and antigens can be used as template molecules. There are two main considerations when designing a molecular printing polymer printed film. Finding the appropriate functional group can form a strong interaction with the template molecule = compound. This strong interaction will be where the complementary template address is the ability to identify in the future. The second is to construct whether the complementary shape of the printed polymer printed film has a binding force, and whether the polymer formed by the polymerization of the monomer can form a hydrophobic interaction force, and a multi-point weak interaction force of hydrogen bonding, so as to identify the ability. Therefore, the choice of different monomers (mostly conjugated double monomers) and the polymer printing film produced by the polymerization environment will have different adhesion characteristics, which can explain that the molecular printing technology is based on the function of the polymer ^ A chemical analysis method derived from the interaction between mechanism and template molecules. The template molecule can be extracted from the insoluble network material, leaving the size, shape and functional group identification sites complementary to the molecular template. The preparation of molecularly printed polymers as stationary phases in a separation system allows selective separation of racemic amino acids or acetophenones. Some systems can use some of the amino acids and winning teams to protect organic solvents. However, the complexity of the protein structure, the variability of the sequence, and the danger of folds make it have page 7 200418987 V. Description of the invention (4) Highly specific group distribution and plate molecules in one molecule to form molecular proteins Molecular imprint, it has many advantages. When the methods of ability are combined with each other, the amount of output signals is difficult. Implementation When the polymer imprint is transferred to make for biological benefits. The current development of the body and protein structure technology has the appearance of low concentration, the use of power, timely detection, and profit measurement, that is, the ability to identify proteins, which can only introduce weak complementary interaction forces through the functions of the three-dimensional space Free radical polymerization in orderly arrangement of functional monomers and molds, low energy configuration, printing polymers. Therefore, after the polymerization is completed, the molecular printed film polymerized by the saccharine or plate is used to maintain the complementary shape and the identified address to those template groups. The success of the brushing technique has been applied to biological and chemical immunoassays that provide functions that have more man-made bond sites than weather antibodies that existed in the past. The manufacture or license to provide a highly discerning sample. At this time, if the phase of the bonded test substance and the molecular identification element can be read on the transducer and become a useful and can be used as a label for interpretation, many examples of purification or separation can be omitted. Try to use the oxytocin (Oxytocin) to brush the template of the membrane to identify the function. Then, using the NS 1 protein as a template for this polymer printed membrane has the same recognition function. It is believed that this will be more helpful for immunoassay analysis techniques, and molecular printed templates can be applied to thermal insulation. Because traditional chemical analysis techniques cannot keep up with living things, there are still many ways to measure the instantaneous reactions in life phenomena in a timely manner. The molecular printed film-biosensor has high specificity, high sensitivity, sensitivity of instant response, and can propose a solution to this problem. Chemical analysis uses the interaction between molecules to identify substances and the background of this technology.

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Dengue virus structurally has 7 segments including non-structural proteins Ng 1, ns2 A, NS2B, NS3, NS4A, NS4B and NS5. Its function is to roughly regulate the growth and replication of the virus', but some are not very clear. In general, the non-structural protein gene has a lower chance of mutation. Dengue can cause antigen-antibody immune response in serology. There are two main parts. One is Nonstructural protein 1 (NS1). One of the coat proteins of prion (Enveiope protein; E protein for short). ), NS1 and E protein are the most in the serum of patients with dengue fever in the early stage of illness. In the present invention, pure NS1 protein is manufactured, and N S1 cores are printed on a piezoelectric crystal by molecular printing technology (MI Ps), and microgravity measurement is performed. The sensitivity of QCM is combined with molecular printing. The ability to recognize forms an excellent sensor that can detect biomolecules. Description of the invention: The present invention uses the method of bio-wafer detection, especially on the coating of the wafer, to use molecular printing technology to polymerize into a polymer printed film with the ability to identify bio-molecules. The purpose of the present invention is to provide a technology capable of manufacturing a biochip by molecular printing technology and applying it to a piezoelectric crystal device to sense biomolecules, especially using a monomer (Acr-Cys-NΗΒη) 2 on a wafer It can be used as an adsorbent and also a linker (1 inker), and it is easy to polymerize with other monomers to form a polymer printed film, which has become a highly stable technology. It is confirmed by the data shown in the following examples that the present invention successfully uses polymer printing

200418987 V. Description of the invention (6) The brush mold has high specificity and high sensitivity to cause changes in the quality on the wafer, and the oscillator (Oscillating circuit) also changes the frequency, so that MIPs-QCM can be used to detect Peptide and NS1 protein of dengue virus. When using a template that is a polymer printed film, 'this M IPs-QCM chip can be used to distinguish this pattern, and when a protein is used as a template for a polymer printed film, this can also be distinguished. A template protein. '' The invention can also detect infectious diseases caused by dengue virus or other viruses, and can be conveniently, time-saving and quickly applied to clinical detection, and more

Due to the mechanization and automation of the process, such biochip products can be commercialized. P Technical Description: The present invention uses molecular printing technology to form a molecularly printed film 'on a wafer by a radical polymerization method to form the ability to identify biomolecules. The piezoelectric wafer used is a quartz plate with two gold electrode quartz wafers in the middle, like a sandwich; but because gold is an inert metal, it is not easy to carry out oxidation reactions on the gold surface and it can resist atmospheric pollution . In addition, gold and sulfur have a strong special interaction, which allows the functional groups of sulfur compounds to form monolayers (SAMs). Example 1: Covering a molecular printed film with 0.5 mg (Acr-Cys-NHBn) 2 (as shown in Formula 1) in 10 ml of aceton i tr i 1 e and a small amount of 0.1 ml DMF mixed uniform solution

Page 10 200418987 V. Description of Invention (7). The wafer was immersed in the solution overnight, so that the disulfide-bonded molecules could be adsorbed from the solution onto the gold surface to form an ordered and positioned single-layer film (SAj | s). The wafer is dried in a drying cabinet. Each prepared 110 // mole of acrylamide, acrylic acid, and N-benzyl-acryl amide early body solution were mixed uniformly in a ratio of 1: 1: 2; the other 15 mg of Oxytocin (as in Example 2), Vasopressin (as in Example 3), partial structure of NS1 (as in Example 4), or NS1 protein (as in Example 5) dissolved in 1 ml of acetonitrile and 20 mmol in a ratio of 6: 4 Ear phosphoric acid solution of ρΗ4.0, and mix well. Use a micro-injection syringe to suck 4 # L of the monomer mixture, place it lightly on the wafer, and then place the wafer in a 20 m 1 vial, and cap the bottle. The vial was placed in a photochemical instrument with an adjusted wavelength of 350 nm for 6 hours, and the monomer solution was subjected to photochemical polymerization. After the polymerization is completed, the crystal moon is taken out, and the surface of the wafer is cleaned with a portion of the acetic acid solvent. Finally, a small amount of pure water is used to clean the remaining solution, and then it is placed in a drying box to completely dry the wafer. The prepared wafer is placed in a jetting tank to perform a jetting reaction. No-flow immunological monitoring was performed using a 0.02 M buffered acid buffer (pH 4.0) as the working solution. The test solution used in the experiment is a solution concentration of! 1 ng / ml of oxytocin (〇Xyt〇cin), 1 mg / ml ~ 1 ng / ml of vasopressin (\ ^ 50 口 『633111), 111 ^ / 1111 ~ 111It / 1111 ~ 31 winning team solution , Angiotensionll II, Angiotensionll, Bradykinin and 0.1 mg / m 1 lng / ml NS1 protein solution were all prepared with the same concentration of phosphate solution.

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Example 2: When oxytocin (such as formula 2) is used as a template for molecular printing, oxytocin flows into the injection flow analysis system at a concentration of 1000 pg / m 1, and the figure 1 is interpreted by the interpretation of the oscillator signal (Picture 1) The change of the frequency of the stomach is caused by oxytocin. It is known that the oxytocin chip has strong sensitivity to oxytocin. At a concentration of 100 pg / ml, it can be detected that the frequency change value is maintained at about 6 Hz. Figure 1 shows the frequency of oxytocin molecules detected by different concentrations of oxytocin solutions. The interaction force of the oxytocin molecular film on the oxytocin molecule can cause the oxytocin molecule to be attracted to the template molecule, thereby forming a fixed frequency value. The higher the concentration, the higher the recognition and binding capacity of the molecular printing template. Its frequency change value can be read from Table 1. Table 1. Oxytocin wafer injection with different concentrations of oxytocin> Frequency change value of cereal fluid to oxytocin molecules Oxytocin concentration (/ ml phosphate buffer) Frequency change value (Hz) 100 pg 6 1 ng 11 10 ng 19 100 ng 22 1 β g 24 10 // g 26 100 g 31 1 mg 58

200418987

Embodiment 3: Similarly, a blood pressure hormone (such as the formula 2 ^ film plate is used to make a subliminin wafer. When two = two f is used for polymer printing, the blood pressure concentration is 100i ′ as a molecular printing The template uses the interpretation of the oscillator signal to draw the picture into the Zhuang flow analysis system, and it is known that the gold dust chip pair is willing. 2) The -1ηπ 7 of the rate change is stronger than that of the blood pressure factor. At a concentration of 100 ºP, the frequency change value of the debt can be measured.

The ability to identify and combine has also improved. Its frequency value can be read from Table 2. The picture shows the identification of blood pressure molecules in the blood pressure solution without undulations. The frequency value detected by the moon force. It is proved again from Fig. 2 that the interaction between the blood pressure molecule molecule and the blood pressure molecule can cause the blood pressure molecule to be attracted to the template molecule, thereby forming a fixed frequency value. The larger the concentration, the molecular printing template Table 1. Frequency changes of blood pressure hormone molecules injected with different concentrations of blood pressure hormone wafers to blood pressure molecules. Blood pressure concentration (/ ml phosphate buffer) Frequency change value (Hz)

100 pg 5 1 ng 7 1 0 ng 10 100 ng 14 1 β g 18 10 μg 22 100 β g 24 1 mg 32 Page 13 200418987 V. Description of the invention (quote) " " " 一 " —- -Based on Figures 2 and 4, it was found that the produced oxytocin and blood pressure hormone-like polymer printing 貘 has a strong ability to recognize 能力 molecules, which can be used as a new embodiment 4: After the membrane's ability to recognize the dendritic virus's N S1 protein (as shown in Formula 4) as a molecularly printed membrane, after the same production process is completed, NS 1 wins crystals are used to test at different concentrations. The amount of change in frequency of NS1 tritium solution, and the test results are as follows

Figure 3 (Figure 3). Figure 3 shows the frequency values detected by the NS1 solution with different concentrations. It was proved once again that the interaction force of the NSi-winning sub-membrane on the NS1 victory can attract 1 peptide to the template molecule and form a fixed frequency value. The higher the concentration, the higher the recognition and binding capacity of the NS1 peptide molecular weight plate. Its frequency value can be read from Table 2. Its frequency change value has excellent sensitivity with increasing concentration.

Page 14 200418987 V. Description of the invention (11) Table III. Frequency change of NS1 win solution with different concentration of NS1 win solution injected with NS1 win solution Wafer concentration of NS1 win solution (/ ml phosphate buffer solution) Frequency change value (Hz ) 1 ng 3 1 〇ng 10 10 0 ng 15 1 β g 33 10 H g 58 1 00 // g 73 1 mg 135 Example 5 ··

Due to the high sensitivity and identification of the NS1 polymer membrane, there is a specific interaction with the NS1 protein in dengue virus serum. The purified virus serum was injected into the NS1 protein to observe the frequency change caused by the specific interaction between the molecular printed membrane and the NS1 protein. From Figure 4, it can be clearly found that the wafer can easily identify the NS1 protein. As the concentration increases, the frequency value increases. The greater the concentration, the higher the recognition and binding power of the molecular printing template of the NS1 team. The frequency value can be read from Table 4 and its frequency change value has an excellent detection effect with increasing sensitivity.

200418987 V. Description of the invention (13) Table 3. Frequency change value of NS1 protein injected with NS1 protein solution of different concentration on NS1 wafer. NS1 concentration (/ ml phosphate buffer) Frequency change value (Hz 1 ng 3 10 ng 6 10 0 ng 10 1 β g 13 1〇β g 27 100 // g 45 0. 5 mg 60 With the above illustration and technical description, the present invention is characterized by the use of molecular printing technology, which can be used as an adsorbent and linker, and It is a monomer (Acr-Cys-ΝηΒη) 2 on the wafer, and it is easy to polymerize with other monomers to form a high-molecular printed film, which has become a highly stable technology. It is established to detect the winning team, protein molecules, and biochemical antibodies. MIPs —QCM sensors have the advantages of high sensitivity, timely detection and simple operation.

Page 16 200418987 Brief description of the diagram Formula 1: The structure of (Acr-Cys-NHBn) 2 (Acr-Cys-NHBn) 2 plays a multi-functional role in this invention. First, it uses the function of the central double-luo bond to adsorb onto the wafer, making it an adsorbent. It is also a cross-linking agent, which can form a connection with the mixed monomer and the wafer. At the same time, the functional group with double and double bonds on the periphery of (Acr-Cys-NHBn) 2 can function as a monomer in the copolymerization reaction. In the structure, the shape of the printed molecules is formed. Formula 2: The structure of the oxytocin molecule. The structure of one win I composed of nine amino acids. In this invention, it is used as a template for the polymer film of the Katsuya sensor. Formula 2: The structure of the vasopressin molecule is a peptide structure composed of nine amino acids. In this invention, it is used as a template for a polymer film of a Suntech sensor. Formula 4: Structural formula of NS1 tritium. The 15 amino acid fragments of the dengue virus NS1 protein were used as templates for the polymer membrane of the trigonometric sensor. The picture shows the ability of the oxytocin wafer to inject the oxytocin solution with different concentrations to identify the oxytocin and the son.

200418987 Brief description of the figure. Figure 2 shows the ability of the blood pressure hormone chip to inject different concentrations of blood pressure solution to identify the blood pressure molecule. Fourth, the identification of NS1 protein by injecting different concentrations of NS1 protein solution for NS1 chip

Claims (1)

200418987 Six, apply for a full-time ® ® ----------- 1 go to species #, and overlay molecular printed film on the wafer to identify biomolecules; an organic compound with an adsorbent and a linker and a monomer Median said, "Single-layer adsorption formation-monolayer" and then add the biomolecules tested by humans. 来 :: A solution of a monomer compound with a heart double bond, which is identified by the polymouth method & Biomolecule-capable polymer printed films. 2. As described in the first item of the patent application, the organic compound with a double bond cross-linking adsorbent and monomer is a derivative containing a photogenic acid structure. 3. As described in the second item of the scope of the patent application, bamboo biology with a photogenic acid structure is a compound containing L-photogenic acid, D-photogenic acid or racemic photogenic acid. 4. As described in the second item of the scope of patent application, the derivatives containing the photoamine structure are (Acr-Cys-NHBn) 2, (Acr-Cys-NH < l)) 2, (Macr-Cys-NHBn) 2, (Macr-Cys-NΗΦ) 2, and other compounds. (Macr is isobutylene) 5. As described in the first item of the scope of patent application, the monomer with a double bond is (Macr-Cys-NHBn) 2, (Macr-AA-NHBn) 2, (Macr-Cys-NΗφ) 2, (Macr-AA-NH Φ) 2, methacrylamide, methacrylic acid, N_benzyl-methacryl amide, ( Acr-Cys — NHBn) 2, (Acr-AA-NHBn) 2, (Acr-Cys-ΝΗΦ) 2, (Acr-AA-ΝΗΦ) 2, acrylamide, acrylic acid, and N-benzyl-acΓylamide. (AA is 20 natural amino acids; φ is benzene) Page 19 200418987 6. Scope of patent application 6. As described in the first item of the scope of patent application, ‘wherein the templates are amino acids, nucleic acids, sugars, lipids, tritium, proteins, antibodies, and antigens. 7. As described in the sixth item of the patent application, where the peptide template is oxytocin 0 8. As described in the sixth item, the peptide template is vasopressin 9, which is the first Among the six items, the winning template is a non-structural winning team of dengue virus. 10. As described in item 6 of the scope of the patent application, the protein molecular printing template is a non-structural protein of dengue virus. 11. As described in the first item of the patent application scope, wherein a single layer is formed by adsorption on the piezoelectric wafer, (Acr-Cys-NHBn) 2 is dissolved in 10 ml of acetic acid and mixed with a small amount of 0.1 l of DMF. Homogeneous solution, then place the solution on the wafer Brother page 20 1 2. As described in the first item of the scope of the patent application, where the solution of the monomeric δ compound with a double bond is a mixture of 1 1 0 " m 〇 1 e, acrylic amine, acrylic acid And N-benzyl-acrylamide monomer solution are mixed uniformly in a ratio of 1: 1: 2; the template molecule is dissolved in 1 ml of acetonitrile and 20 mmol in a ratio of 6: 4 200418987 6. Application patent scope Ear pH 4 · In the acid filling solution of 〇 ', the two will be mixed uniformly at a ratio of 1 u 0 1 3. As described in item 范围 of the scope of the patent application, the polymerization method is: light irradiation in a photochemical instrument with a wavelength of 3 50 nm Hours, the monomers are formed into polymer, polymer, or heated to 50 ~ 100. C completes the polymerization. ΰ 1 4. A method of “identifying biodiversity by coating molecular printed film on a wafer”, which uses a single layer of (Acr-Cys-NHBn) 2 compound on a piezoelectric wafer, and then uses a double bond Acrylamide and propionate formation br ^: ryil: ide ^^ ^ ^ ^ n Learn 1 law to make it with ...