Chapter 4. Gold Nanoparticle Antibody Conjugates for Use in Competitive Lateral Flow Assays

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1 Chapter 4 Gold Nanoparticle Antibody Conjugates for Use in Competitive Lateral Flow Assays Julian Bailes, Samantha Mayoss, Phil Teale, and Mikhail Soloviev Abstract Gold nanoparticles (GNPs) are widely used in a variety of biomedical diagnostic assays and for imaging. Their popularity stems from key properties such as their low toxicity and high extinction coef fi cients, as well as straightforward synthesis methods that allow GNPs to be produced quickly and inexpensively. Here we describe the use of GNPs for visual detection in a lateral fl ow assay using benzodiazepine af fi nity assay to illustrate the methods Key words: Lateral fl ow, Immunochromatography, Test strip, Gold nanoparticles, Au nanoparticles, Colloidal gold, Antibody conjugation 1. Introduction 1.1. Lateral Flow Assays Immunochromatographic assays are often designed to provide easy-to-use point-of-care test kits. Lateral fl ow or strip tests are one such example, embodying speed and simplicity over high multiplexity ( 1, 2 ). These tests provide rapid results and are relatively inexpensive to produce making them well suited for mass markets and routine assays compared to lab-based assays such as SPR- and QCM-based technologies. Such kits are usually stable for long durations across a wide range of environments and conditions and can be made compatible with all major sample matrices including urine, saliva, serum, plasma, and whole blood. Although immunochromatographic assays were fi rst described in the 1960s, it wasn t until Unipath s Clearblue pregnancy test ( ) was released in the late 1980s that they saw their fi rst commercial release. Clearblue provided rapid results for just a single analyte, Mikhail Soloviev (ed.), Nanoparticles in Biology and Medicine: Methods and Protocols, Methods in Molecular Biology, vol. 906, DOI / _4, Springer Science+Business Media, LLC

2 46 J. Bailes et al. Fig. 1. Schematic diagram of assembled lateral flow components. The sample pad (A) is where the test sample is fi rst deposited and regulates fl ow rate into the subsequent conjugate pad (B) where it mixes with a labeled reference reagent (represented here as a gold antibody conjugate) that has been temporarily dried in place. Sample and labeled reference reagent diffuse along a porous membrane (D) coming into contact with capture (E) and control (F) lines (represented here as protein drug conjugates and antibodies, respectively). An absorbent pad (G) completes the lateral fl ow strip and serves to maximize the sample volume entering the membrane test strip, thus fl ushing through unbound detection particles, reducing background and increasing sensitivity. All components are assembled on an adhesive backing layer (C) and usually housed in a plastic test cassette with defined sample application and test result windows. human chorionic gonadotropin (hcg) ( 3, 4 ) and while pregnancy tests remain one of the few mass produced ( 5 ), and arguably the best known of lateral fl ow assays, the technology has been applied in many sectors, from healthcare screening ( 6, 7 ), to veterinary and agricultural applications ( 8 ), to biodefense and detection of infectious agents ( 9 ). Unipath s Clearview product range alone now offers point-of-care diagnostics for pregnancy and ovulation, STDs, infectious disease, respiratory disease, and coagulation/cardiac conditions. Lateral fl ow assays are compatible with direct and competitive immunoassay formats, although in practice competitive formats are preferred because they do not require labeling or spotting of the sample prior to use, which is not at all practical in most situations where the tests are to be used and defeats the main attractions of such tests, their simplicity and speed. In their simplest form, lateral fl ow tests consist of a band of capture antibodies (marked E in Fig. 1 ) immobilized on a porous membrane strip (marked D in Fig. 1 ). The assay relies on the diffusion of samples along the length of the strip by capillary forces towards target-speci fi c binding sites. A porous membrane is typically a strip of nitrocellulose although nylon, polyethersulfone, polyethylene, and fused silica can be used. Normally lateral fl ow test devices include a number of other components such as a sample pad (marked A in Fig. 1 ) which can serve several critical purposes including regulating fl ow rate into the subsequent conjugate pad, and fi ltration when separation of

3 4 Gold Nanoparticles for Lateral Flow Assays 47 Fig. 2. Photograph of completed sample run shown in fully assembled lateral fl ow test device complete with designated sample window, and with top casing removed. Capture line (deposited at 13 mm from origin of membrane) and control line (at 16 mm) both clearly visible indicating sample is negative. plasma from whole blood is required. A conjugate pad (marked B in Fig. 1 ) is typically included and acts as a zone for labeled reference reagent that is dried in place but becomes mobile as the sample defuses through it. A control line (marked F in Fig. 1 ) located after the capture line is also a common feature and provides con fi rmation that the assay has successfully run to completion. An absorbent pad (marked G in Fig. 1 ), typically cellulose fi ber, completes the lateral fl ow strip and serves to maximize the sample volume entering the membrane test strip, thus fl ushing through unbound detection particles, reducing background and increasing sensitivity. All components are attached to a backing layer (marked C in Fig. 1 ) and usually housed in a plastic test cassette with de fi ned sample application and test result windows (see Fig. 2 ). For direct assay strips the visual presence of a capture band upon completion of the assay indicates a positive sample, whereas in the competitive format reduced color intensity or disappearance of the capture line entirely is indicative of a target analyte s presence in the sample. For a more detailed consideration of lateral fl ow devices and materials, see ref. ( 10 ). The production and reproducibility of lateral fl ow tests is simpli fi ed and increased by using quali fi ed equipment such as the Biodot platforms ( ) that can quantitatively dispense reagents onto membranes in bulk and perform precise cutting of batch preparations into individual strips Gold Nanoparticles While the original Clearblue test used blue latex particles for visual detection, the past 20 years have seen many alternative labeling and visualization techniques employed, including liposomes that contain reporter agents such as colored, fl uorescent or bioluminescent dyes ( ), carbon ( 14, 15 ), selenium ( 16 ), upconverting

4 48 J. Bailes et al. phosphors ( ), quantum dots ( 20 ), electrochemiluminescent particles ( 21 ), and silica particles ( 22 ). More recently, the use of gold nanoparticles (GNPs) for colorimetric detection in lateral fl ow was reported for testing of troponin 1 ( 23 ), bacterial contaminations ( 24 ), explosives ( 25 ), and disease detection ( 26 ). Among the many reasons for their popularity in the fi eld of medical diagnostics and biological imaging is the fact that they are nontoxic, possess extinction coef fi cients far greater than that of organic dyes ( 27 ), and can be produced simply and inexpensively in almost any lab. Synthesis involves chemical reduction of tetrachloroauric acid (HAuCl 4 ) solution with sodium citrate ( 28 ). Controlling the amount of reducing solution determines the size of the particles, which in turn determines their color. Although the citrate method is the most commonly used, other methods of synthesis have been reported over the years ( ). Important considerations for any detection particles to be used in lateral fl ow are that the population can remain monodisperse and consists of particles with a consistent, spherical shape so as to ensure consistency of migration through the porous membrane. Proteins readily adhere to the nanoparticle surface due to the combination of electrostatic attraction, covalent bonding, and hydrophobic interactions. If insuf fi cient ligand molecules are present on the surface of the nanoparticle then aggregation may occur when in the presence of buffers. Benzodiazepines are a group of drugs characterized by a benzene ring fused with a diazepine ring and are agonists of the receptor for the neurotransmitter gamma-aminobutyric acid (GABA), which is responsible for regulating neuronal excitability and muscle tone. Here we describe the use of GNPs for visual detection in a benzodiazepine lateral fl ow assay. We illustrate the lateral fl ow preparation and assembly methods using anti-benzodiazepine GNP conjugates as an example. 2. Materials 2.1. Antibody Conjugation to GNPs Use deionized water for all buffers unless stated otherwise. 1. Potassium carbonate buffering reagent (K 2 CO 3 ): make 0.2 M solution in water. 2. Colloidal gold: 40 nm (see Notes 1 and 2). 3. ph paper test strips (ph range 0 14). 4. Hydrogen peroxide (H 2 O 2 ): 30 % solution in water. 5. Mouse anti-benzodiazepine monoclonal antibody: unconjugated, clone BEN1 (Biogenesis, now part of AbD Serotec), protein-a puri fi ed in phosphate buffered saline (PBS) buffer (ph 7.2) with 0.05 % sodium azide (see Note 3).

5 4 Gold Nanoparticles for Lateral Flow Assays Bovine serum albumin (BSA): make 10 % (w:v) solution in water. 7. Benchtop refrigerated centrifuge capable of 10,000 g at 4 C and 2 ml microcentrifuge tubes. 8. Resuspension solution: 1 % sucrose, 0.1 % sodium azide in water Preparation of Oxazepam Lateral Flow Strips 2.3. Lateral Flow Assays 1. Release agent: 0.5 % Tween-20, 0.5 % human serum albumin, in water. 2. Glass fi ber conjugate pad strips: mm ( G041 from Millipore). 3. Dispensing solution: 20 mm PBS, 5 % methanol, 0.1 % lactose (see Note 4). 4. Sodium chloride (NaCl): make M solution in water. 5. Capture reagent: 2.5 mg/ml oxazepam-3-hemisuccinate/ BSA conjugate (OX-3HS-BSA) in dispensing solution (see Note 5). 6. Control reagent: 1 mg/ml Goat anti-mouse IgG, whole molecule (Sigma) in M sodium chloride. Dilute 1/10 in dispensing solution to a fi nal concentration of 100 μ g/ml before use. 7. PBS: 0.01 M phosphate buffer, M potassium chloride, and M sodium chloride, ph 7.4, at 25 C. 8. Nitrocellulose membrane: Hi-Flow Plus 240 SN-HF, mylarbacked nitrocellulose, capillary fl ow rate 240 s per 4 cm (Millipore UK Ltd.) (see Note 6). 9. Dispensing robot for dispensing control and capture line reagents onto membrane: BioDot XYZ3000 Platform with BioJetQuanti 3000 (BioDot Ltd) (see Note 7). 10. Cutting module for cutting batch product into individual test strips: BioDot CM4000 Guillotine Cutting Module (BioDot Ltd.) (see Note 7). 11. Blocking solution: 1 % dried milk powder, 0.1 % sodium azide in dih 2 O. 12. SureWick Cellulose fi ber sample pad strips: mm and mm, plastic backed (Millipore). 13. Adhesive backing card: mm (Millipore). 14. Plastic test cassettes for lateral fl ow assembly, such as shown in Fig. 2 (see Note 8). 1. Diazepam: 1 mg/ml solution of diazepam in methanol. 2. PBS: as in item 7, Subheading 2.2 (see Note 9). 3. Prepare fi ve serial dilution of diazepam in PBS ranging from 1 to 10,000 ng/ml in tenfold increments. Make fresh before use.

6 50 J. Bailes et al. 3. Methods 3.1. Antibody Conjugation to GNPs 3.2. Preparation of Oxazepam Lateral Flow Strips This section describes the conjugation of BEN1 monoclonal antibody to GNPs. The methods are easily adaptable for use with any other immunoglobulins. 1. Adjust the ph of colloidal gold solution and BEN1 monoclonal antibody solution to ph 9.5 using 0.2 M K 2 CO 3, check ph using ph paper (see Note 10). 2. Add 30 % H 2 O 2 to the colloidal gold solution to a fi nal concentration of 0.1 % (see Note 11). 3. Dilute mouse anti-benzodiazepine monoclonal antibody in water to a fi nal concentration of 50 μ g/ml (see Note 12). 4. Mix the antibody and colloidal gold solutions in a ratio of 1:5, respectively, by slowly adding the gold to the protein while mixing briskly (see Note 13). 5. Mix the solution gently at room temperature for min. 6. Add 10 % BSA to the antibody gold conjugate to a fi nal concentration of 1 %. Mix gently at room temperature for 15 min. 7. Purify the conjugate by centrifugation at 4 C for at least 1 h at 10,000 g (see Note 14). 8. Carefully remove and discard supernatant that contains proteins (see Note 15). Do not disturb the pellet. 9. Resuspend the gold conjugate in resuspension solution (see Note 16). 10. Determine absorbance at 520 nm as a measure of the conjugate concentration (see Note 17). 11. Store nanoparticles at 4 C until use (add NaN 3 to fi nal concentration of 0.02 % if conjugate is to be stored for over 48 h). 1. Take a precut glass fi ber strip of mm. Soak the strip in the release agent for 5 min and dry it at 37 C for 2 h (see Note 18). 2. Soak the dried, treated glass fi ber strip in anti-benzodiazepine monoclonal antibody gold conjugate (from step 11, Subheading 3.1 ) (see Note 19). Dry at 37 C for 2 h. 3. Dispense capture and control lines onto nitrocellulose membrane at 13 and 16 mm from origin (marked E and F, respectively, in Fig. 2 ). Allow membranes to dry for 10 min at 37 C (see Note 20). 4. Block membranes by submersion in blocking solution for 30 min at room temperature (see Note 21).

7 4 Gold Nanoparticles for Lateral Flow Assays Rinse membranes in dh 2 O (3 10 s) and then dry for 30 min at room temperature. 6. Assemble cellulose fi ber sample pads, treated membrane strip, and treated glass fi ber strip on 300 mm wide adhesive plastic backing card such that there is a 2 mm wide overlap between individual components. Leave to set for 2 h at room temperature (see Fig. 1 ). 7. Cut the assembled card into 5 mm wide strips and place each into a plastic test cassette (see Fig. 2 ). 8. Store desiccated at room temperature until use Lateral Flow Assays 1. Apply 3 4 drops ( μ L) of diazepam spiked PBS or blank PBS control sample onto sample window of test cassettes (see Fig. 2 ) and allow the test to run for 15 min (see Note 22). 2. Allow 15 min after adding the sample before reading the assay results. Colored band may form at capture line and will form at control line. Appearance of a band at the control line con fi rms successful test run. Appearance of two bands (capture and control lines) indicates a negative sample, while the partial or total loss of intensity in color of the capture band indicates a positive sample. 4. Notes 1. We used BBI (British Biocell International) but many other suppliers are currently available, e.g., Aldrich (Sigma-Aldrich) and Fitzgerald (Fitzgerald Industries International). 2. Although readily available from a large number of commercial suppliers, GNPs can be produced relatively easy, see e.g., ( 28 ) for the details of nanoparticle manufacturing. 3. Polyclonal antisera can be used but requires puri fi cation (e.g., by ammonium sulfate precipitation of IgG followed by immunoaf fi nity puri fi cation) prior to GNP conjugation. 4. Methanol ( 5 %) is added to improve binding of proteins to nitrocellulose (>5 % may damage proteins), and lactose is added to improve stability. Proteins bind best at or near their isoelectric point and so ph 4.5 is used for the BSA drug conjugate in this instance. For consistency, this solution is also used for control reagents despite not being optimized for the antibodies. 5. To maximize assay sensitivity ensure that the concentration of conjugate used as capture reagent is as low as possible while still yielding a visible signal.

8 52 J. Bailes et al. 6. When choosing which nitrocellulose membrane to use there are a number of important factors to consider, namely slower membrane fl ow rates increase the time reagents spend in proximity, thus increasing sensitivity, and that the membrane pore size must be at least ten times the diameter of any particles used as labels. For a more detailed consideration of lateral fl ow materials, see ref. ( 10 ). 7. While manual apparatus (pipettes and scissors) can be used when preparing later fl ow test strips, its use will compromise consistency. For best results use specialized automated equipment, such as BioDot XYZ3000 Platform and BioDot CM4000 Guillotine Cutting Module to improve reproducibility and output. 8. A variety of similar cassettes are commercially available, e.g., from Millipore ( ), Diagnostic Consulting Network ( ), and Advanced Microdevices ( ). 9. It is important to develop and optimize the assay for the particular matrix it is intended for, e.g., pooled plasma. Flow characteristics and assay performance will be affected by the rheology of the matrix used. Also consider the color of the sample when choosing a label to work with in order not to compromise visual signal detection. 10. The ph of the colloidal gold solution and the protein to be conjugated should be adjusted to 0.5 ph units above the isoelectric point of the protein. This ensures the overall negative charge on the proteins and their adhesions will not change the surface charge of the NPs, thus avoiding aggregation. Do not use a ph meter when adjusting the ph of colloidal gold to avoid damaging the electrode. 11. H 2 O 2 is used to break down residual tannic acid. 12. To determine protein binding capacity of GNPs, prepare a series of BSA dilutions ranging from 1 μ g/ml to 10 mg/ml. Add 100 μ L of each of BSA dilution to individual 500 μ L aliquots of colloidal gold and incubate the samples at 25 C with constant, gentle agitation. Following the 30 min incubation add 600 μ L aliquots of 20 % NaCl to individual GNP BSA samples. The GNP BSA sample with the lowest BSA content (100 μ L or 1 mg/ml BSA per 500 μ L of the GNP preparation) where no color change is observed contains suf fi cient protein for total coverage of the colloidal gold present. This ratio of total protein to GNPs should be achieved by the conclusion of the blocking stage (step 6, Subheading 3.1 ) in order to ensure complete coverage of the GNPs. The above assays may be scaled down to reduce reagents usage.

9 4 Gold Nanoparticles for Lateral Flow Assays Fig. 3. Absorption spectra of gold nanoparticles. Dashed line shows absorption spectrum of nonderivatized NPs. Absorption maximum 522 nm. Solid line same nanoparticles after derivatization with protein (bovine albumin in this example). Absorption maximum 528 nm. 13. Slowly adding the gold to the protein results in a more stable conjugate and minimizes the risk of fl occulation (aggregation of gold particles). 14. Unconjugated protein will remain in the supernatant. The conjugate may form a two-phase pellet consisting of looser particles (the conjugate) and very dense, black particles (aggregates of gold particles). Aggregates may be an indication that the amount of protein used in the blocking step was insuf fi cient to achieve complete coverage of the nanoparticles, in which case the conjugation procedure should be restarted. 15. Remove the supernatant carefully as conjugate may go into suspension very easily. 16. Sucrose in the buffer will facilitate the resuspension and will prevent aggregation of conjugate and improve stability during storage. 17. The reported extinction coef fi cients for gold NPs vary (e.g., M 1 cm 1 for 16 nm gold NPs ( 32 ) or M 1 cm 1 for 12 nm gold NPs ( 33 ) ). For simplicity one can assume to be 10 8 M 1 cm 1, meaning that peak absorbance of 0.1 will correspond to 1 nm NP concentration. Peak wavelength will depend on the nanoparticle size. One can therefore estimate nanoparticle diameter using this formula: λ max = d where d is nanoparticle diameter in nanometers ( 34 ). Absorbance spectrum of protein gold conjugate should show a redshift (see Fig. 3 ).

10 54 J. Bailes et al. 18. Treating the glass fi ber with surfactants, polymers, or proteins prior to application of gold conjugate ensures it accepts and releases the majority of the label applied to it. 19. The glass fi ber pad ( mm, G041 from Millipore) holds 45 μ L/cm 2. Ensure a suf fi cient concentration of gold conjugate is used. The amount of antibody absorbed will eventually be redissolved in the sample applied to the strip. Hence the fi nal working antibody concentration will depend on both the amount loaded and the sample volume. 20. When dispensing capture and control reagents remember that protein will bind to nitrocellulose where it comes into contact with it, not where the buffer might subsequently appear to spread. These positions are suggested as guidelines for a 60 mm test strip (including sample, conjugate, and absorbent pads). Flow rate is a function of the distance from the origin and so altering the location of capture and control lines will in turn affect assay kinetics and sensitivity. Be sure that your chosen positions fall within the viewing window of any test cassette you intend to use. 21. Slowly slide the membrane into the blocking solution at a shallow angle to reduce the risk of air becoming trapped in the three-dimensional structure of the membrane. 22. Orientation and temperature should be standardized, i.e. test cassette placed on fl at surface and test run at room temperature. References 1. Qian S, Bau HH (2003) A mathematical model of lateral fl ow bioreactions applied to sandwich assays. Anal Biochem 322(1): Qian S, Bau HH (2004) Analysis of lateral fl ow biodetectors: competitive format. Anal Biochem 326(2): May K (1991) Home tests to monitor fertility. Am J Obstet Gynecol 165(6): Chard T (1992) Pregnancy tests: a review. Hum Reprod 7(5): Cole LA (2011) The utility of six over-thecounter (home) pregnancy tests. Clin Chem Lab Med 49(8): Wu AH, Smith A, Christenson RH et al (2004) Evaluation of a point-of-care assay for cardiac markers for patients suspected of acute myocardial infarction. Clin Chim Acta 346(2): Rossi AF, Khan D (2004) Point of care testing: improving pediatric outcomes. Clin Biochem 37(6): Liao JY, Li H (2010) Lateral fl ow immunodipstick for visual detection of a fl atoxin B-1 in food using immuno-nanoparticles composed of a silver core and a gold shell. Microchim Acta 171: Peruski AH, Peruski LF Jr (2003) Immunological methods for detection and identi fi cation of infectious disease and biological warfare agents. Clin Diagn Lab Immunol 10(4): Posthuma-Trumple GA, Korf J, van Amerongen A (2009) Lateral fl ow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey. Anal Bioanal Chem 393(2): Gussenhoven GC, van der Hoorn MA, Goris MG et al (1997) LEPTO dipstick, a dipstick assay for detection of Leptospira-speci fi c immunoglobulin M antibodies in human sera. J Clin Microbiol 35(1):92 97

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