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Tips and Tricks for Colloidal Gold Test Strip-Development

Tips and Tricks for Colloidal Gold Test Strip-Development

richardjgray
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Tips and Tricks for Colloidal Gold Test Strip-Development

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  1. Tips and Tricks for Colloidal Gold Test Strip Development CD What is colloidal gold? Colloidal gold is a colloidal suspension of gold nanoparticles synthesized by the reduction of chloroauric acid (HAuCl4) into gold ions under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, and tannic acid. It consists of a basic crystal nucleus (icosahedron formed by 11 gold atoms) and a surrounding double ion layer (the inner layer is a negative ion layer, AuCl2-; the outer layer is the positively charged H+). Due to the electrostatic action, the gold particles repel each other and suspend into a stable colloidal state, forming a negatively charged hydrophobic colloid solution, hence the name colloidal gold. Colloidal gold has high electron density and can be conjugated with a variety of biological macromolecules. It has become a commonly used non-radioactive tracer in immune labeling technology after fluorescein, radioisotope, and enzyme. The particle size of colloidal gold is generally between 1-100nm, which can be uniformly and stably dispersed in the liquid, showing small spherical particles or large oval particles. The outermost layer of colloidal gold particles can absorb a large number of positive ions and can be uniformly dispersed in the solution. Because the surface of colloidal gold is negatively charged, it can be labeled with positively charged antigens and antibodies by electrostatic interaction. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  2. What is colloidal gold? As shown in Figure 1, the sample is applied to one end of the test strip on the sample pad. The adsorption sample pad is impregnated with buffer salts and surfactants to make the sample suitable for interaction with the detection system. The sample pad ensures that the analyte present in the sample can bind to the capture reagent of the conjugate and bind to the membrane. The processed sample migrates through the binding pad, which contains antibodies against the target analyte and is bound to color or fluorescent particles (the most commonly used are colloidal gold and latex microspheres). The sample migrates along the test strip to the detection area along with the conjugated antibody bound to the target analyte. This is a porous membrane (usually composed of nitrocellulose). Specific biological components (mainly antibodies or antigens) are immobilized on the membrane to provide a place to react with the analyte bound to the conjugated antibody. The identification of the sample analyte will produce an appropriate response on the test line, while the response on the control line indicates that the liquid flowing through the test strip is correct. The results represented by the lines appearing with different intensities can be evaluated with the naked eye or using a dedicated reader. In order to test multiple analytes at the same time under the same conditions, antibody test lines for different analytes can be fixed in an array. The liquid flowing through the device is due to the capillary force of the strip material, and in order to maintain this movement, an absorbent pad is attached to the end of the strip to absorb excess reagent and prevent liquid backflow. Sample and Buffer Colloidal Gold Labeled Antibody Test Line Control Line Sample Pad Conjugate Pad Membrane Backing Adsorbent Pad Test Strip C T Figure 1. Schematic diagram of Sandwich colloidal gold test strip. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  3. Advantages and disadvantages of colloidal gold test strip Advantages Disadvantages Rapid: all reactions can be completed in 15 minutes Easy to operate: convenient for personal self-test or hospital testing Low cost: no special equipment is required Good stability: the effective period of the reagent can be up to two years or more  The sensitivity is lower than that of ELISA and chemiluminescence methods Prone to false positive and false negative HOOK effect may occur       Colloidal gold test strip components The colloidal gold test strip is composed of sample pad, conjugate pad (containing colloidal gold), membrane (e.g. NC film), and absorbent pad, all are connected to each other from top to bottom and affixed to the backing card (usually a PVC base plate). There is a testing line and a control line on the NC film. Sample pad • It is where the test sample is dropped. The sample pad has filtering and buffering effect on the test sample, and reduces the ionic strength or pH of the sample from interfering the test result. It is typically composed of a woven mesh or cellulose fiber. Regardless of the material, the sample pad should exhibit consistent absorbency, thickness, and density so that a uniform wicking rate ensures the reproducibility of the analysis. The sample pad should also show low protein binding to avoid loss of analyte. Conjugate pad • The colloidal gold-labeled antibody is fixed here by drying for the reaction between the antibody and the antigen and is usually composed of non-woven glass fiber. This antibody is key in controlling the performance of colloidal gold test strips. The conjugate pad should exhibit low non-specific binding so that the detection reagent does not remain. Pre-treatment may be necessary to increase wettability, reduce non-specific interactions, and control pH. It is important that the conjugate pad has a consistent bed volume to ensure that the amount of detection reagent in each test strip remains constant. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  4. Membrane • In the colloidal gold test, the membrane is considered the most important element. Nitrocellulose is the most commonly used material, although cellulose acetate, polyvinylidene fluoride (PVDF), charged modified nylon, and polyethersulfone (PES) can also be used. It is essential that the thickness of the film keeps consistent to avoid any mechanical damage. In order to avoid the variability of the assay, the membrane must remain completely flat when it is being striped with antibodies. Therefore, it may be preferable to choose a nitrocellulose membrane with a non-porous backing film. If the test strip is to be placed in a plastic cassette, it is important to ensure that the thickness of the plastic liner is the same. Otherwise, the test strip may be compressed. The film is pre-coated with test line and control line. The test line is the primary reading for diagnosis and consists of immobilized proteins that can bind to colloidal gold to produce signals related to the presence of analytes in the sample. The control line contains an affinity ligand that captures colloidal gold conjugates with or without analytes in the solution to confirm that the test is working properly. Absorbent pad • The absorbent pad makes the liquid sample flow to one side through capillarity, and at the same time drives the movement of the colloidal gold-labeled antibody on the adsorption pad, thus reacting with the antigen on the test line. Assembling procedures There are generally two assembly methods, namely batch processing and in-line assembly. Batch processing allows the use of low-cost equipment and is suitable for relatively low production volumes, but its disadvantage is that it requires large amount of manual labor and is prone to product variability. In the in-line production process, the test strips are machined in continuous rolls, which can increase output and significantly reduce product variability. Regardless of the assembly method chosen, it is essential to control the environmental conditions (temperature and relative humidity) during the pad drying, dispensing, and strip assembly processes. Protein dispensing is usually conducted at 18-25°C and the relative humidity should be 40-60%, while strip assembly and storage are usually performed at 18-25°C and the relative humidity should be <20%. Fluctuations in these conditions may bring additional variability to the test strip. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  5. Synthesis of colloidal gold Colloidal gold is usually synthesized by reduction of chloroauric acid (HAuCl4), using some sort of stabilizing agents. The first step is to dissolve HAuCl4 and then stir the solution quickly and add a reducing agent at the same time to reduce the Au3+ ions to neutral gold ions. Commonly used reducing agents are white phosphorus, ascorbic acid, trisodium citrate, and tannic acid-trisodium citrate. Gold particles with different diameters can be obtained by changing the ratio of chloroauric acid to reducing agent in the reaction system. However, the diameter of gold particles prepared by the first two reducing agents is not uniform, and the latter two are commonly used at present. Commercial colloidal gold is also a good choice. Trisodium citrate reduction method (reference process shown below) • 1. 10% chloroauric acid of 10 µL is added to 100 mL deionized and distilled water (in a glass bottle). 2. Boil the solution. 3. While stirring the solution violently with a stirring rod, quickly add 4 mL of freshly prepared 1% trisodium citrate aqueous solution. 4. Continue to boil the solution under stirring. The color of the solution will turn blue in about 5-7 minutes and then red. In another 2-3 minutes, the reaction will reach the endpoint and the solution will be red-orange. 5. Boil for another 5 minutes, then cool and transfer the solution to a clean spiral lid glass bottle. If keep refrigerated and avoid light, the colloidal gold will be stable. Notes: a) Do not use the chloroauric acid that has not been sealed in a glass vial. b) Clean the vial thoroughly and rinse with deionized distilled water. c) The chloroauric acid solution is stable for years if kept refrigerated and protected from light. d) For 19 nm colloidal gold, add 3 mL of 1% trisodium citrate; for 12.5 nm, add 6 mL of 1% trisodium citrate. e) Care should be taken not to let the solution evaporate, which will change the concentration of the components. The siliconized 250 ml volumetric flask works well. Alternatively, a reflow device can be used. Conjugation of colloidal gold Under alkaline conditions, the surface of colloidal gold particles is negatively charged and can bind firmly to the positively charged groups carried by antigens/antibodies. This binding has no significant effect on the biological activity of the conjugated antigens/antibodies. pH and ionic strength are the main reasons that affect the adsorption of colloidal gold and antigen/antibody. The size of colloidal gold particles, molecular weight and concentration of protein also affect the adsorption of protein. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  6. Protocol • Example: conjugation of Biotin (MW = 244.3 Da) to DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles, 10 nm (Cat. GFC-3K-10). 1. Prepare a 10 mM MES buffer and adjust pH to 5.0 with NaOH. 2. Prepare a stock solution of 100 mg/mL of Biotin in dimethyl sulfoxide. 3. Wash 2 filter units with deionized water. 4. Bring 2 mL of DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles, 10 nm in each of the 2 filter units. 5. Centrifuge at 1500xgav for 10 minutes. 6. Add 3 mL of 10 mM MES buffer, pH 5.0 per filter unit, mix and centrifuge at 1500xgav for 15 minutes. 7. Remove the concentrated DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles from the filter units and adjust the collected volume to 4 mL with 10 mM MES buffer, pH 5.0. 8. Immediately before use, prepare a 100 mM EDC solution in 10 mM MES buffer, pH 5.0. 9. Add 80 µL of 100 mM EDC in MES buffer to 4 mL of DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles and mix well. 10. Incubate for 5 minutes. 11. Immediately before use, prepare a 100 mM sulfo-NHS solution in MES buffer. Adjust the pH to 5.0. 12. Add 80 µL and incubate for 30 minutes. 13. Add 20 µL Biotin stock solution. Incubate for 2 hours. 14. Centrifuge at 1500xgav for 10 minutes using the filter units. Repeat this step twice. 15. Collect the concentrated biotinylated conjugate. Dilute to OD520nm = 1.0 in PBS, 0.1% BSA, 15 mM NaN3, pH 7.4. Notes: a) Determination of the pH for the optimal reaction of each antibody often requires multiple experiments. b) The centrifugal speed and time vary for gold particles of different particle size. c) Both citrate and carbonate are easily displaced by other molecules or ligands, such as those with terminal amine or sulfhydryl groups. Therefore, these surfaces are ideal for passive binding of antibodies to the surface of nanoparticles. d) Carbonate is smaller than citrate, so it is easier to be displaced, resulting in a higher surface load of physically adsorbed proteins. e) DiagNanoTM Carboxyl Gold Nanoparticles provide a highly negatively charged surface and a further functionalized chemical handle. The carboxyl surface can be used to covalently bind molecules with free amines (such as antibodies) to the surface of nanoparticles. f) Covalent conjugates are more stable than conjugates prepared by passive adsorption because the amide bond is permanent and the antibody will not dissociate over time. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  7. g) The covalent coupling process does not depend on the isoelectric point of the antibody, eliminating the pH optimization process, saving time, and reducing costs. h) The amount of antibody required for the covalent conjugate per unit particle is usually less than that required for passive adsorption. i) Compared with passive adsorption, covalent conjugation is more reproducible and easier to control the amount of antibody attached to each particle. Commercial colloidal gold • Cat. No. Product Name BG-40 DiagNanoTM Gold Nanoparticles, 40 nm BG-60 DiagNanoTM Gold Nanoparticles, 60 nm GFC-3K-40 DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles, 40 nm GFC-5K-40 DiagNanoTM Carboxyl, PEG5000 Gold Nanoparticles, 40 nm GFC-3K-60 DiagNanoTM Carboxyl, PEG3000 Gold Nanoparticles, 60 nm GFC-5K-60 DiagNanoTM Carboxyl, PEG5000 Gold Nanoparticles, 60 nm GCK-40 DiagNanoTM Gold Nanoparticle Covalent Conjugation Kit, 40 nm GCK-60 DiagNanoTM Gold Nanoparticle Covalent Conjugation Kit, 60 nm GPK-40 DiagNanoTM Gold Nanoparticle Passive Conjugation Kit, 40 nm GPK-60 DiagNanoTM Gold Nanoparticle Passive Conjugation Kit, 60 nm GPK-LF DiagNanoTM Gold Nanoparticle Passive Conjugation Kit, Lateral Flow NGC-150 DiagNanoTM Carboxyl Gold Nanoshells, 150 nm Factors that may affect the quality of the developed test strips The isotypes of antibody • In general, the IgG1 isotype antibody is the best for colloid gold conjugation. Although other isotypes have some limitations, we often use IgG2a and IgG2b, occasionally use IgM and IgA. The purity of proteins • When the purity of the target antibody is low, it will cause competitive conjugation of colloid gold to the target antibody and other miscellaneous proteins, thus affecting the reaction performance of in vitro diagnostic reagents, such as its specificity, reaction signal, background value, and so on. The use of a serum-free medium can greatly remove the impurities in the serum medium, while the protein purification method can further remove most of the impurities in the antibody. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  8. Protein purification method • Method Problems Solution Recombinant antigen/antibody is usually labeled with his-tag which uses imidazole to elute. Thus, the antigen/antibody diagnostic materials purified by nickel column always contain a large amount of imidazole. However, even a small amount of imidazole can make colloidal gold agglomeration and aggregation. Nickel column purification The serum purified by ammonium sulfate or DEAE contains a large number of miscellaneous proteins that can competitively bind to the binding sites on the surface of colloidal gold, resulting in strong electrolytes and high salt components. It affects the results and reduces the sensitivity of the assay. Ultrafiltration or dialysis: For the dialysate, choose the buffer with which pH deviates from the isoelectric point of the target protein as far as possible. If the isoelectric point of the protein is uncertain, it is recommended to use 20 mM Tris-HCL or 20 mM Tris-glycine as dialysate, which is suitable for most antigen and antibody raw materials. Ammonium sulfate protein precipitation or DEAE purification If the mouse peritoneal serum antibody is purified by protein A or protein G column chromatography and used as colloidal gold labeling raw material, because the antibody raw material contains a large amount of non- specific mouse IgG, there will be a large number of non-specific colloidal gold conjugates, thus reducing the sensitivity of the assay. Protein A/G purification Storage buffer • For better storage, some substances such as glycerol and sucrose may be added as protective agents. However, these agents will adversely affect colloidal gold conjugation because of their strong hydrophilicity. Ideal antigen and antibody storage solutions are 10 mM/20 mM PB (pH7.4-8.0), 10 mM/20 mM PBS (pH7.4-8.0), 20 mM Tris-HCL (pH8.0-9.0), 20 mM Tris-glycine (pH8.0-9.0). In addition, an appropriate amount of preservatives can be added, such as 0.1%-0.5% sodium azide. Tel: 1-631-633-6938 Email: info@cd-bioparticles.com

  9. FAQs 1. If the colors of C and T lines are not consistent, how to judge the test results? • For the competitive method, as long as C and T lines are colored within a specified period of time, regardless of whether the color is consistent or not, it can be determined as negative. The result is positive if the T line does not show color. • For the colorimetric method, if the T line is deeper than C line, the result is negative. If the T line is lighter than C line or T line does not show color, the result is positive. • For the sandwich method, as long as the C and T lines are colored within a specified period of time, the result can be read positive. The darker the T line, the stronger the positive. 2. Can I use water or other liquids as negative samples in the process of using colloidal gold test cards? No. Because the composition of the test sample is very complex, using water as a negative control has no reference significance. In addition, it is also easy to cause false positive or missed detection. 3. Troubleshooting Troubleshooting Causes Solutions Increase the sample amount or add some auxiliary liquid Pretreat the sample to remove the sediment Use a new test strip  Not enough sample added The sample is too sticky The test strip is invalid  The colloid gold test is not working     When testing whole blood, a large number of samples were precipitated, and the background of the NC membrane was reddish, which affected the interpretation of the results The NC film of the test strip was seriously damaged or broken Use hemolytic whole blood samples  Replace a new test strip and re-test Try to use a fresh whole blood sample    The detection time is too short or reading the result too early Not enough sample added, or too much auxiliary liquid diluted the sample Concentration of the tested substance in the sample is too low pH value of the sample is too high or too low The test strip was exposed to moist, and its activity decreased or deactivated  Read the results at the time required by the instructions, and it is recommended to wait more time for weak or negative samples Correctly add sample or auxiliary liquid as required by the instructions Confirm with other detection methods    High rate of missed detection     (continued) Email: info@cd-bioparticles.com Tel: 1-631-633-6938

  10. The test strip has high sensitivity The sample contains special interfering substances or has been contaminated Read the result after the specified time Replace a new test strip and re-test and read the results at the specified time. Confirm the test results with other testing methods to ensure that the samples are not contaminated    High false positive rate of test results   Creative Diagnostics offers a comprehensive list of coated, functional and conjugated gold nanoparticles by precisely surface engineering. We provide gold nanoparticles functionalized with active groups such as NHS, carboxyl, amine and hydroxyl. Conjugated nanoparticles with a variety of biological ligands are also available for your colloid gold test strip development. For more information, view our website: www.cd-bioparticles.com Email: info@cd-bioparticles.com Tel: 1-631-633-6938 Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Fax: 1-631-938-8221 5

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