A method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials

Abstract

This invention belongs to the field of biosensing and detection technology, and discloses a method for preparing and applying a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials. A MCOF@Au nanocomposite material with both fluorescence signal and magnetic response capabilities was prepared, combined with an MTDN-assisted signal amplification strategy. APT was modified onto the surface of MCOF@Au via Au-S bonds to construct an MCOF@Au / APT complex. The S-chain labeled with CdTe QDs bound to MTDN to obtain an MTDN / S fluorescent signal probe. MCOF@Au / APT and MTDN / S were incubated to form a functionalized MCOF@Au / APT / MTDN / S complex. In the presence of the target acrylamide (AA), APT specifically binds to AA, causing MTDN / S to detach from the precipitate. After magnetic separation, MTDN / S enters the supernatant, and the fluorescence signal of the precipitate is detected. The fluorescence signal of CdTe QDs at 620 nm decreased, while the fluorescence signal of MCOF@Au remained unchanged at 510 nm. Based on changes in fluorescence intensity, quantitative detection of AA can be achieved. Compared with other fluorescence sensors used for AA detection, the prepared ratiometric fluorescent aptamer sensor has the advantages of high specificity and good stability.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, specifically to a method for preparing and applying a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials. Background Technology

[0002] Acrylamide (AA) is widely found in various fried and baked foods. It is a byproduct of high-temperature food processing and has been proven to have potential carcinogenic effects on humans. Long-term intake poses irreversible potential risks to human health. AA exposure routes mainly include skin contact, inhalation, and dietary intake, with dietary intake being the most significant and critical route of human exposure. Given the widespread presence of AA in the human food chain, developing efficient, accurate, and easy-to-use AA detection technologies is crucial for ensuring food safety and protecting public health.

[0003] Currently, traditional methods for detecting ammonia (AA) mainly include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA). While these methods offer advantages in accuracy and sensitivity, they also suffer from drawbacks such as expensive equipment and time-consuming detection processes, making them unsuitable for real-time on-site detection. Therefore, researchers have explored various combinations of chemical and biological sensing technologies as rapid and economical alternatives for detecting trace amounts of AA, such as colorimetry, electrochemical methods, and fluorescence methods. Among these, fluorescent aptamer sensors, with their high sensitivity, fast response speed, and ease of operation, have become an important tool for AA detection.

[0004] In recent years, reported fluorescence detection methods for AA have all used a single wavelength fluorescence signal as the output. Such sensors are highly susceptible to environmental or experimental conditions, easily resulting in false positives or false negatives, thus limiting the accuracy and sensitivity of quantitative measurements. To improve sensor accuracy, ratiometric fluorescence sensors simultaneously acquire fluorescence signals from two different emission peaks and perform quantitative analysis based on their signal ratio. Compared to traditional single-wavelength emission fluorescence sensors, ratiometric fluorescence sensors have self-calibration characteristics, effectively avoiding false positives and false negatives, and providing more stable fluorescence signals. Furthermore, developing highly sensitive and efficient separation methods is crucial for the fabrication of biosensors. Based on this, this invention fabricates a core-shell structured magnetic MCOF@Au, utilizing its fluorescence signal and magnetic response characteristics as the sensor's internal reference signal and separation method, respectively. Combined with a DNA tetrahedron (MTDN)-assisted signal amplification strategy, the sensor's sensitivity is enhanced.

[0005] This invention addresses the problems of existing detection technologies, such as poor selectivity, low sensitivity, and false positives or false negatives, by constructing a novel ratiometric fluorescent biosensor. First, nucleic acid aptamers are used to specifically recognize the target AA, enhancing detection specificity. Second, MTDN is introduced to amplify the signal, improving detection sensitivity. Finally, magnetic MCOF@Au is used to provide a stable and reliable internal reference signal and magnetic separation capability for the system. Based on this invention, the ratiometric fluorescent sensing strategy overcomes the problems of the existing technologies while being more sensitive, economical, simple, and stable, thus facilitating its widespread application. Summary of the Invention

[0006] This invention designs a method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials. A method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials, comprising the following steps: (1) Preparation of cadmium telluride quantum dots (CdTe QDs): First, sodium borohydride and tellurium powder were dissolved in water and quickly sealed. After the reaction, a transparent purple precursor solution was obtained. At the same time, inorganic cadmium compounds and sulfides were dissolved in water and stirred for a certain period of time. Then, the pH of the solution was adjusted with NaOH, and nitrogen gas was introduced to remove oxygen. Then, under nitrogen protection, the prepared precursor solution was quickly injected into the container. The solution immediately changed from colorless to orange under vigorous stirring. Subsequently, the solution was refluxed until the color turned dark red. The product was washed several times with a mixture of isopropanol and ultrapure water, and then redispersed with ultrapure water before storage.

[0007] (2) Preparation of multifunctional MCOF@Au: Organic carboxylates and inorganic iron salts were dissolved in ethylene glycol and mixed in an ultrasonic bath to form a homogeneous solution. After reacting for a certain time, the solution was cooled to room temperature. The product was washed several times with deionized water, and the solid product was dried to obtain Fe3O4. Aromatic dialdehyde, aromatic triamine, 1,4-dioxane and butanol were mixed uniformly. Then Fe3O4 and acetic acid were dispersed in the solution, and acetic acid was added. After the reaction, the product was cooled to room temperature and collected with a magnet. After washing three times, the product was dried under vacuum to obtain MCOF. MCOF was dissolved in water, and then gold salt and citrate were added and stirred. Then inorganic metal hydride salt solution was quickly added and mixed evenly. The product was washed several times with deionized water, collected by magnetic separation, and dried overnight to obtain multifunctional MCOF@Au nanocomposite material.

[0008] (3) Preparation of ratiometric fluorescent aptamer sensors: T1, T2, T3, and T4 were placed in a PCR instrument, annealed, and then gradually cooled to form a stable MTDN structure. CdTe QDs solution was then incubated with the S chain overnight, followed by the addition of MTDN solution to obtain the MTDN / S complex. APT was incubated with MCOF@Au to obtain the MCOF@Au / APT complex. The MCOF@Au / APT complex was then incubated with the MTDN / S complex to obtain the functionalized MCOF@Au / APT / MTDN / S complex. When the target acrylamide (AA) was present in the system, magnetic separation was performed after incubation, the supernatant was removed, and Tris-HCl buffer solution was added to 200 μL. The fluorescence intensity in the precipitate was detected. The fluorescence intensity change was measured using a fluorometer to establish a curve relating the fluorescence signal response value to the target concentration.

[0009] Further specifying, in step (1), the inorganic cadmium compound is one or more of CdCl2·2.5H2O, CdCl2, etc.; the sulfide is one or more of sulfur monochloride, MPA, sulfur, etc.; the stirring time in water is 5 min; the pH value is adjusted to 9; and the reflux time is 15 h.

[0010] Further specifying, in step (2), the organic carboxylate is one or more of anhydrous sodium acetate, sodium benzoate, sodium citrate, etc.; the inorganic iron salt is one or more of FeCl2, FeCl3·6H2O, Fe2(SO4)3, etc.; the reaction temperature and time are 200℃ and 12 h, respectively; the aromatic dialdehyde is one or more of 2,5-dimethoxy-terephthalaldehyde, terephthalaldehyde, isophthalaldehyde, etc.; the aromatic triamine is one or more of 1,3,5-tris(4-aminophenyl)benzene, 1,3,5-tris(4-aminophenyl)triazine, 1,3,5-tris(4-aminophenyl)acetylene, etc.; the reaction time and temperature are 70℃ and 72℃, respectively. h; the gold salt is one or more of tetrachloroauric acid, potassium gold cyanide, etc.; the citrate is one or more of sodium citrate, potassium citrate, etc.; the inorganic metal hydride salt is one or more of sodium borohydride, potassium borohydride, lithium borohydride, etc.; the stirring temperature and time are 37℃ and 30min, respectively.

[0011] Further specified, in step (3), the concentration of DNA strands T1, T2, T3, T4, APT, and S is 1~2 μM, the volume used is 5~10 μL, and the incubation time is 0.5~2 h; the incubation time is 30~120 min.

[0012] Further specifying, in step (3), the sequence of T1 is 5'- CGA GGA TTT GCC GTT TCC GGTTTT TTT AGA AGA GCC GCC ATA GTA TTT TTA GCT ACA TTG TCT-3'; the sequence of T2 is 5'- TCA ACT GCC TGG TGA TTT TTT TTG TAG CTA AAA ATG CGA GG GTC CAA TAC-3'; the sequence of T3 is 5'- TCA ACT GCC TGG TGA TTT TTT TAA ACG ACA CTA CGT GGG AAT CTA CTATGG CGG CTC TTC-3'; the sequence of T4 is 5'- TCA ACT GCC TGG TGA TTT TTT TTT CAG ACTTAG GAA TGT GCT TCC CAC GTA GTG TCG TTT GTA TTG GAC CCT CGC ATT TTT AGC TACA-3'; the sequence of the S chain is 5'- ATC ACC AGG CAG TTG ATT TTT TTT T-3' where the 5' end is modified with NH2.

[0013] Compared with the prior art, the present invention has the following significant advantages: 1. The present invention prepares a core-shell structured magnetic MCOF@Au, which not only provides a stable and reliable internal reference fluorescence signal for the system, but also enables efficient separation in complex matrices due to its magnetic response characteristics.

[0014] 2. This invention combines an MTDN-assisted signal amplification strategy to enhance the signal strength of CdTe QDs and improve the sensitivity of the sensor.

[0015] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the fabrication method of a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials.

[0017] Figure 2 The fluorescence detection results of the sensor constructed in Embodiment 1 of the present invention before (dashed line) and after (solid line) addition of AA are shown.

[0018] Figure 3This is the standard curve for AA detection constructed by the sensor in Embodiment 1 of the present invention.

[0019] Figure 4 This is a standard curve for detecting AA in biscuit and potato chip matrices using a sensor constructed according to Example 1 of the present invention.

[0020] Figure 5 The sensor constructed in Embodiment 1 of the present invention exhibits selectivity for AA in the presence of other interfering substances. Detailed Implementation

[0021] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0022] A method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials, the implementation method of which is as follows: Figure 1 As shown.

[0023] The specific steps are as follows: (1) Preparation of CdTe QDs First, 36 mg of sodium borohydride and 24 mg of tellurium powder were dissolved in 1 mL of water and quickly sealed. The mixture was reacted at 4 °C for 12 h, yielding a transparent purple NaHTe precursor solution. Simultaneously, 86 mg of CdCl₂·2.5H₂O and 75 μL of MPA were dissolved in 50 mL of water and stirred for 5 min. The pH of the solution was then adjusted to 9 with 1 M NaOH, and nitrogen gas was purged for 30 min to remove oxygen. Next, under nitrogen protection, 1 mL of the prepared NaHTe precursor solution was rapidly injected into the container. The solution immediately changed from colorless to orange upon vigorous stirring. The solution was then refluxed for 15 h until the color turned deep red. The product was washed several times with a mixture of isopropanol and ultrapure water, redispersed with 2 mL of ultrapure water, and stored at 4 °C.

[0024] (2) Preparation of multifunctional MCOF@Au: 2.88 g of anhydrous sodium acetate and 1.08 g of FeCl3·6H2O were dissolved in 40 mL of ethylene glycol and a homogeneous solution was formed in an ultrasonic machine. The reaction was carried out at 200 °C for 12 h and then cooled to room temperature. The product was washed several times with deionized water and the solid product was dried at 60 °C to obtain Fe3O4. 2,5-Dimethoxytetraphenylcarbamate (8.7 mg), 1,3,5-tris(4-aminophenyl)benzene (10.5 mg), 1,4-dioxane (2 mL) and butanol (2 mL) were mixed evenly. Then Fe3O4 (12 mg) and acetic acid (0.05 mL) were dispersed in the solution, and 0.45 mL of acetic acid (12 M) was added. The reaction was carried out at 70 °C for 72 h. The product was collected using a magnet after cooling to room temperature, washed three times with acetone and THF, and then vacuum dried for 12 h to obtain MCOF. 5 mg of MCOF was weighed and dissolved in 10 mL of water, followed by the addition of tetrachloroauric acid (0.25 mL 0.1 M) and sodium citrate (0.25 mL 0.1 M), and stirred for 5 min. Then, sodium borohydride solution (0.425 mL 0.1 M) was rapidly added, and the mixture was stirred at 37 °C for 30 min. The product was washed several times with deionized water, magnetically separated, and dried overnight at 60 °C to obtain the multifunctional MCOF@Au.

[0025] (3) Preparation of ratiometric fluorescent aptamer sensor: 2 μL of 100 μM APT was added to 10 μL of TCEP and shaken at 37℃ for 1 h. Then MCOF@Au was added and incubated at 37℃ for 2 h to obtain MCOF@Au / APT complex. Then 10 μL of 1 μM T1, T2, T3 and T4 were placed in a PCR instrument and heated to 95℃ for denaturation for 10 min, and then gradually cooled to form stable MTDN. 200 μL of prepared CdTe QDs solution, 10 μL of 0.1 M EDC and 10 μL of 0.1 M NHS were mixed and reacted at 37℃ for 3 h. Then 10 μL of 10 μM S chain was added and incubated at 37℃ overnight. Then MTDN solution was added and incubated at 37℃ for 2 h to obtain MTDN / S complex. The prepared MCOF@Au / APT and MTDN / S solutions were reacted at 37 °C for 2 h to obtain the functionalized MCOF@Au / APT / MTDN / S complex. Then, buffer solutions without and containing AA were added, and the mixture was incubated at 37 °C for 30 min. Subsequently, magnetic separation was performed, the supernatant was removed, and Tris-HCl buffer solution was added to a final volume of 200 μL. The fluorescence intensity in the precipitate was detected. The excitation wavelength was set to 330 nm, and the fluorescence intensity at emission wavelengths of 510 nm and 610 nm was recorded using a fluorescence spectrophotometer.

[0026] (4) Establishment of standard curve: Add 10 μL of AA standard solution of different concentrations to step (3) to obtain sample detection solutions of different gradients. After incubation, different fluorescence signals are obtained. The logarithm of AA concentration is used as the abscissa, and the ratio of the fluorescence signal response difference at 620 nm with and without AA at 510 nm is used as the ordinate to perform linear fitting and establish the standard curve of the sensor for AA.

[0027] (5) Establishment of standard curves in biscuit and potato chip matrix: 10 μL of AA solution in biscuit and potato chip matrix of different concentrations was added to step (3). After incubation, different fluorescence signals were obtained. The logarithm of AA concentration was used as the abscissa, and the ratio of the fluorescence signal response difference at 620 nm with and without AA at 510 nm was used as the ordinate to perform linear fitting and establish the standard curve of AA in the matrix by the sensor.

[0028] like Figure 2 The image shows the fluorescence detection results of the sensor constructed in Embodiment 1 of the present invention before (dashed line) and after (solid line) the addition of AA.

[0029] like Figure 3 As shown, this is the standard curve for AA detection by the sensor constructed in Embodiment 1 of the present invention.

[0030] like Figure 4 As shown, this is the standard curve of AA in the sensor biscuit and potato chip matrix constructed in Example 1 of the present invention. Example

[0031] A method for fabricating and applying a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials, the practical application of which includes the following steps: To verify the specific recognition of AA by the newly prepared ratiometric fluorescent biosensor, AA standard was added to Tris-HCl buffer solution to achieve a AA concentration of 50 nM in the sample. Standard solutions of seven other interfering substances (acrylic acid, caffeine, ascorbic acid, glycine, aspartic acid, glucose, and starch) were prepared using Tris-HCl buffer solution, each with a concentration of 100 nM. The detection system constructed in Example 1 was used to detect the above seven different interfering substance standard solutions and their mixtures with AA. The detection results are as follows: Figure 4 As shown, this demonstrates that the method of the present invention has good selectivity for AA. Example

[0032] A method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials is described, and its practical application includes the following steps: (1) Food sample processing: The powdered biscuit and potato chip samples were dispersed in water (200 mg / mL) and sonicated for 30 min. Then, 2.5 mL of n-hexane was added, and the mixture was shaken in a constant temperature shaker for 15 min. After centrifugation at 4000 rpm for 15 min, the operation was repeated to complete the defatting process. The resulting precipitate was treated with Carrez I and II (1 mL each) to disrupt the emulsion and precipitate proteins and carbohydrates. After centrifugation at 4000 rpm for 15 min, the supernatant was obtained, filtered through a 0.45 μm microporous membrane, and diluted with Tris-HCl for later use. The food extract was obtained using the standard addition method.

[0033] (2) Sample detection: Take 10 μL of food extract and measure the fluorescence signal according to step (3) of Example 1. Substitute the signal into the standard curve to obtain the concentration of AA in the sample.

[0034] (3) When using biscuits as food samples for determination, 0.1 times and 10 times the standard amount of AA were added to potato chips, respectively, based on the addition amount of 5 nM. Take 10 μL of sample solution and measure the fluorescence signal according to step (3) of Example 1. The AA concentration in the sample was obtained by adding the standard solution in the biscuit and potato chip matrix detected in Example 1. Each sample was measured three times and the average value was taken. The average recovery rate of the sensor was calculated to be 94.34%~118.38%.

[0035] The novel ratiometric fluorescence sensor developed has been validated to exhibit good specificity, stability, reproducibility, and repeatability in the detection of amino acids (AA). This sensor enables quantitative analysis of AA in complex samples.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0037] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials, characterized in that, Includes the following steps: (1) Preparation of fluorescent signal probes: cadmium telluride quantum dots (CdTe) QDs: Sodium borohydride and tellurium powder were dissolved in water and quickly sealed. After the reaction, a transparent purple precursor solution was obtained. At the same time, inorganic cadmium compounds and sulfides were dissolved in water and stirred. Then, the pH of the solution was adjusted with NaOH, and nitrogen gas was introduced to remove oxygen. Then, under nitrogen protection, the prepared precursor solution was quickly injected into the container. The solution immediately changed from colorless to orange under vigorous stirring. Then, the solution was refluxed for a certain period of time until the color turned dark red, and stable CdTe QDs were obtained as fluorescent signal probes. (2) Preparation of multifunctional MCOF@Au nanocomposite material: Organic carboxylate and inorganic iron salt were dissolved in ethylene glycol and formed into a homogeneous solution in an ultrasonic machine. After reacting at a fixed temperature for a period of time, the solution was cooled to room temperature to obtain Fe3O4. Aromatic dialdehyde, aromatic triamine, 1,4-dioxane and butanol were mixed uniformly. Then Fe3O4 and acetic acid were dispersed in the solution. Acetic acid was added and the product was cooled after reacting for a certain period of time to obtain magnetic covalent organic framework MCOF. Taking advantage of the large surface area and good stability of MCOF, gold salt and citrate were added and stirred to react. Then inorganic metal hydride salt solution was quickly added and stirred evenly to obtain multifunctional MCOF@Au nanocomposite material with both fluorescence signal and magnetic response capabilities. (3) Preparation of ratiometric fluorescent aptamer sensor: First, DNA strands T1, T2, T3, and T4 were placed in a PCR instrument and annealed, gradually decreasing to a low temperature to form a stable DNA tetrahedral structure; then, CdTe QDs solution was incubated with DNA strand S overnight, and then the DNA tetrahedral structure solution was added, and incubation was carried out to obtain a DNA tetrahedral / S complex; DNA strand APT was incubated with MCOF@Au to obtain an MCOF@Au / APT complex; then, the MCOF@Au / APT complex was incubated with the DNA tetrahedral / S complex to obtain a functionalized MCOF@Au / APT / DNA tetrahedral / S complex; when the target acrylamide AA was present in the system, AA bound to DNA strand APT, causing the DNA tetrahedral / S to detach from the complex, and magnetic separation was performed, the DNA tetrahedral / S entered the supernatant, the supernatant was removed, and Tris-HCl buffer solution was added to 200 μL. The fluorescence intensity in the precipitate was detected; By measuring the change in fluorescence intensity using a fluorescence meter, a curve relating the fluorescence signal response value to the concentration of the target analyte was established.

2. The method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials according to claim 1, characterized in that, In step (1), the inorganic cadmium compound is one or more of CdCl2·2.5H2O, CdCl2, etc.; the sulfide is one or more of sulfur monochloride, MPA, sulfur, etc.; the stirring time in water is 5 min; the pH is 9; and the reflux time is 15 h.

3. The method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials according to claim 1, characterized in that, In step (2), the organic carboxylic acid salt is one or more of anhydrous sodium acetate, sodium benzoate, sodium citrate, etc.; the inorganic iron salt is one or more of FeCl2, FeCl3·6H2O, Fe2(SO4)3, etc.; the fixed temperature and reaction time are 200℃ and 12 h, respectively; the aromatic dialdehyde is one or more of 2,5-dimethoxy-terephthalaldehyde, terephthalaldehyde, isophthalaldehyde, etc.; the aromatic triamine is one or more of 1,3,5-tris(4-aminophenyl)benzene, 1,3,5-tris(4-aminophenyl)triazine, 1,3,5-tris(4-aminophenyl)acetylene, etc.; the reaction time is 72 h; the gold salt is one or more of tetrachloroauric acid, potassium gold cyanide, etc.; the citrate is one or more of sodium citrate, potassium citrate, etc.; the inorganic metal hydride salt is one or more of sodium borohydride, potassium borohydride, lithium borohydride, etc.; and the stirring time is 30 min.

4. The method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials according to claim 1, characterized in that, In step (3), the concentration of DNA strands T1, T2, T3, T4, APT, and S is 1~2 μM, and the volume used is 5~10 μL; the incubation time is 30~120 min.

5. The method for fabricating a ratiometric fluorescent aptamer sensor based on multifunctional MCOF@Au nanomaterials according to claim 1, characterized in that, In step (3), the sequence of T1 is 5'- CGA GGA TTT GCC GTT TCCGGT TTT TTT AGA AGA GCC GCC ATA GTA TTT TTA GCT ACA TTG TCT-3'; the sequence of T2 is 5'- TCA ACT GCC TGG TGA TTT TTT TTG TAG CTA AAA ATG CGA GG GTC CAA TAC-3'; the sequence of T3 is 5'- TCA ACT GCC TGG TGA TTT TTT TAA ACG ACA CTA CGT GGG AAT CTACTA TGG CGG CTC TTC-3'; the sequence of T4 is 5'- TCA ACT GCC TGG TGA TTT TTT TTT CAGACT TAG GAA TGT GCT TCC CAC GTA GTG TCG TTT GTA TTG GAC CCT CGC ATT TTT AGCTAC A-3'; and the sequence of the S chain is 5'- ATC ACC AGG CAG TTG ATT TTT TTT T-3' where the 5' end is modified with NH2.

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