Rather than detecting the amount of an enzyme through traditional lateral-flow immunoassays, this technology detects only active enzymes and has the potential for detecting a plethora of enzymes and enzyme inhibitors involved in bond cleavage and bond formation.
The lateral-flow immunochromatographic assay (LFIA) format has achieved considerable commercial success, as demonstrated by strong sales of pregnancy and drugs-of-abuse tests.1-4 Because of its simplicity, low cost, and user-friendliness, LFIA is particularly attractive for point-of-care and over-the-counter tests. Although LFIA has been primarily used for qualitative detection, several new technologies capable of generating quantitative measurements have emerged.
Such technologies include colorimetry, conventional fluorescence, time-resolved fluorescence, phosphorescence, magnetic field measurements, surface-enhanced Raman, and up-converting luminescence.5-11 In fact, due to their proven robustness, some of these technologies have received regulatory approval for application in clinical diagnostics.
A lot of work has also focused on improving the detection of nucleic acids in LFIA. Several types of molecular probes and detection methods have been combined with lateral-flow assays to enhance the analysis of DNA and RNA.12 In addition, LFIA has been used for the simultaneous detection of several analytes. Although the throughput is limited, this detection platform can achieve multiplexing of analytes.13
LFIA does have limitations, such as the ability to determine only the amount of antigens and haptens in a sample. For detecting enzymes, LFIA cannot differentiate between active enzymes and denatured forms. In some cases, such differentiation is desirable and essential for clinical diagnosis. For example, active proteases from pathogens such as hyphal yeasts are believed to be responsible for vaginal yeast infections.14,15 Monitoring the enzymatic activity of proteases would also benefit the monitoring of a drug therapy’s efficacy.
Although tremendous efforts have been made in developing and commercializing LFIA for detecting a large number of analytes, minimal work has been done in adapting the lateral-flow assay format for detecting enzyme activity. This article discusses efforts to integrate an enzyme reaction with a lateral-flow assay technology. In addition to detecting enzyme activity, these assays also provide a simple method to screen and identify enzyme inhibitors. Compared with conventional enzyme assays, lateral-flow enzyme assays require no instrumentation. Finally, these assays are simple, rapid, and inexpensive, yet provide high sensitivity.
Methods and Materials
Surface-functionalized particles and magnetic particles, including carboxylated and streptavidin-modified blue particles (SA-BP, 0.3 µm), were obtained from Bangs Laboratories. Sulfo-NHS-LC-biotin was acquired from Pierce Biotechnologies. Bovine serum albumin (BSA), β-casein, streptavidin, anti-biotin antibody, and Streptomyces griseus (SG) protease were bought from Sigma-Aldrich. Phosphate buffered saline (PBS; 50 mM sodium phosphate, 100 mM sodium chloride, pH=7.2), sodium borate, carbodimide, and ethanolamine were purchased from Polysciences Inc. Nitrocellulose membranes, supporting cards, cellulose samples, and wicking pads were obtained from Millipore Inc.
Preparation of Casein-MP. 25 mg of 0.35 µm magnetic particles (MP) with carboxylic acid surface groups were washed once with borate buffer and once with PBS. The separation of the magnetic particles from the supernatant was accomplished by a magnet separator. The washed particles were suspended in 1 ml PBS buffer, and 28.8 mg carbodimide in 1 ml PBS buffer was added. The mixture was gently shaken for 15 minutes. The particles were washed four times with a borate buffer (pH=8.5, 0.1 M).
The washed particles were suspended in 1 ml borate buffer, and two mg β-casein in 1 ml borate buffer was added. The resulting mixture was gently shaken at room temperature overnight. The particles were washed three times with water, and suspended with one ml of 50 mM ethanolamine solution in water. The mixture was shaken for 30 minutes. The particles were washed five times with water and suspended in 2 ml borate buffer. These particles will be referred to as casein-MP for the remainder of this article.
Preparation of Casein-BP. The procedure is the same as covalent attachment of β-casein to magnetic particles, except that carboxylated blue latex particles (0.3 µm) were used to replace the carboxylated MP. Separation and washing were accomplished by centrifugation. These particles will be referred to as casein-BP for the remainder of this article.
Preparation of Biotin-Casein-MP and Biotin-Casein-BP. Four mg of casein-MP, or casein-BP, in 300 µl borate buffer was added to 1 mg sulfo-NHS-LC-biotin in 200 µl borate buffer (50 mM, pH=8.4). The mixture was gently shaken for four hours and washed five times using tris buffer (20 mM, pH=7.2). The particles were suspended in 1 ml tris buffer for storage and were designated as biotin-casein-MP or biotin-casein-BP.
Preparation of Biotin-BSA. 500 mg BSA in 9 ml borate buffer was added to 300 mg sulfo-NHS-LC-biotin in 1 ml borate buffer (50 mM, pH=8.4). The mixture was allowed to react for three hours at room temperature. The mixture was dialyzed five times in PBS buffer. The biotinylated BSA is designated as biotin-BSA.
Preparation of Type I Strips. A hi-flow 120 nitrocellulose membrane on a plastic support card was striped with a line of 5 mg/ml biotin-BSA in water to form Capture Zone I. A similar line of polylysine solution (50 mg/ml) in water was printed to form Capture Zone II. All striping was conducted using a dispenser by Kinematic Automation. The printed card was dried overnight at 37° C.
A cellulose wicking pad was laminated to the end of the nitrocellulose membrane, close to Capture Zone II, with a 5-mm overlap. A conjugated pad and a sample application pad were laminated with the side of the nitrocellulose membrane, close to Capture Zone I. For the conjugate pad, a 10-cm-long strip of glass fiber pad material was loaded with a solution of 50 µl of 1% SA-BP, 200 µl 20% sucrose, 100 µl 2% tween 20, and 250 µl 20 mM tris buffer (pH=7.2), followed by drying at 37° C for four hours. The card was cut into 4-mm-wide strips.
Preparation of Type II Strips. A hi-flow 120 nitrocellulose membrane on a plastic support card was striped with a line of 2 mg/ml anti-biotin antibody in water to form Capture Zone I and with a line of polylysine solution (50 mg/ml) in water to form Capture Zone II. The membrane was dried overnight at 37° C. A cellulose wicking pad was laminated to the end of the nitrocellulose membrane, close to Capture Zone II, with a 5-mm overlap. The card was cut into 4-mm wide strips.
Detection of Protease Using Magnetic Substrate Conjugates. Each of the six samples containing 80 µg of biotin-casein-MP in 60 µl 20 mM tris buffer (pH=7.2) was spiked with a different amount of metalloprotease, ranging from 0.02 to 4.0 µg per sample. A control sample containing 200 µg of deactivated metalloprotease was included. The samples were incubated at room temperature for 20 minutes, and the magnetic particles were removed by a magnet separator. 20 µl of the resulting supernatant from each sample was applied to the sample pad of each type I device. The strips were developed for 30 minutes (see Figure 3).
Detection of Metalloprotease Using Non-Magnetic Substrate Conjugates. Five samples, containing 0.3 µg of biotin-casein-BP in 40 µl 20 mM tris buffer (pH=7.2), 1% tween 20, were spiked with 0-40 ng of metalloprotease. The samples were incubated for 20 minutes at room temperature. A type II strip was inserted into each sample for development. The strip was developed for 30 minutes (see Figure 5).
|Figure 1. Schematic representation of lateral-flow enzyme assays. S and P represent substrate and product, respectively. LFIA stands for lateral-flow immunoassay.|
Results and Discussion
The general process for lateral-flow enzyme assays is depicted in Figure 1. The method combines an enzyme reaction with LFIA. An enzyme substrate is catalyzed by an enzyme to generate one or more products. The reaction may cleave a chemical bond, form a new bond, or result in a conformational isomerization. If the test is intended to screen for an enzyme inhibitor, the inhibitor will also be present in the samples. The lateral-flow test strip can be used after the enzyme reaction is completed, or the enzyme reaction can be integrated into the lateral-flow test strip, which will need a certain mechanism to control reaction time and condition. For example, a lateral-flow test strip may be coupled with a microfluidic device to control reaction time and conditions.
Theoretically, LFIA can be constructed to detect the disappearance of a substrate, the appearance of products, or both simultaneously, provided antibodies are available. However, such antibodies may not exist, especially for small molecular substrates such as a short peptide. In these cases, it is desirable to tag the substrate with specific binders and/or probes. Depending on the substrates and enzymes, different assay formats can be constructed.
|Figure 2. Process of a lateral-flow enzyme assay using magnetic particles.|
Magnetic Lateral-Flow Enzyme Assays
Figure 2 depicts an assay format that uses a magnetic particle, a substrate, and a specific binder I (B1) for detecting an enzyme, which involves a bond cleavage to release a product. An enzyme substrate is covalently linked to a magnetic particle and B1 to form a magnetic particle-based substrate conjugate. When an enzyme cleaves the substrate conjugate to release B1 from the magnetic particle, the free B1 can be separated from both digested and intact substrate conjugates by a magnetic field, and detected by a lateral-flow test strip.
To demonstrate the principle, metalloprotease from SG was used as a model system. β-casein is a good substrate for the metalloprotease. Biotin-casein-MP was prepared as an enzyme substrate.
The amide bonds of β-casein on biotin-casein-MP are cleaved by the protease to release some or all of the biotin moieties from the MPs. The MPs are removed by a magnet, and the biotin molecules in the solution can be detected on type I lateral-flow test strips. The lateral-flow test strip for detecting biotin can be universally used for assaying various enzymes as long as the enzyme reaction releases biotin from a substrate conjugate.
|Figure 3. Detection of protease by a lateral-flow enzyme assay using magnetic particles.|
Figure 3 shows the results for detecting metalloprotease in a series of samples spiked with a different amount of metalloprotease. Without active protease, all biotin molecules are attached to the MPs and are removed from the solution by a magnet, and therefore no biotin remains in the solution. When the solution is applied on a type I lateral-flow test strip, all the SA-BP particles released from the conjugate pad were captured in Capture Zone I by the immobilized biotin-BSA to show a strong blue line (200 µg deactivated). Capture zone II showed almost no blue signal.
In the presence of 0.02-0.2 µg of active protease, some of the biotin molecules were released from the MPs. The released biotin molecules competed for the SA-BP so that fewer SA-BP were able to be captured by biotin-BSA in Capture Zone I, resulting in a weaker signal. SA-BPs saturated with released biotin molecules passed through Capture Zone I and bound to the polylysine in Capture Zone II to produce a strong signal. The positively charged polylysine in Capture Zone II can capture any particles with a negative charge. Therefore, the color density of Capture Zone I decreased as the enzyme activity increased, while the color density of Capture Zone II increased. When the amount of the protease was one or four µg, all the biotin molecules were released from the substrate conjugates into the solution. In this case, Capture Zone I showed no blue line, while Capture Zone II formed a strong blue line.
|Figure 4. Process of a lateral-flow enzyme assay using colored particles|
Particle Lateral-Flow Enzyme Assays
Figure 4 depicts another lateral-flow enzyme assay format that uses a non-MP such as a colored or fluorescent particle. An enzyme substrate is covalently tagged with a specific binder I (B1) and a colored particle to make a substrate conjugate. Without an enzyme, the particles are captured by a second binder (B2, specific for B1) that is immobilized in Capture Zone I on a type II lateral-flow device to show a strong color signal. Capture Zone II should show either no signal or a weak signal if the capturing capacity of Capture Zone I for the substrate conjugate is more than the total amount of the substrate conjugate.
In the presence of an enzyme that cleaves the substrate, some or all of the B1 will be released from the particles. The released B1 can flow faster than the substrate conjugates to bind with the B2 in Capture Zone I. Furthermore, the digested or partially digested substrate conjugates will have fewer B1 moieties; therefore, they will have less or no chance of being captured by the B2 in Capture Zone I to produce a weaker signal or no signal. The digested or partially digested substrate conjugates pass through Capture Zone I and are captured by the polylysine in Capture Zone II to show a stronger signal.
|Figure 5. Detection of protease by a lateral-flow enzyme assay using colored particles|
To demonstrate the principle of the assay format in Figure 4, biotin-casein-BP was prepared as a substrate conjugate for metalloprotease from SG. Figure 5 shows the developed test strips for analyzing samples spiked with 0-40 ng of metalloprotease. In the absence of metalloprotease, the substrate conjugates are captured in Capture Zone I of a type I strip to show a strong signal. Capture Zone II showed no signal because all the substrate conjugates were captured in Capture Zone I.
In the presence of 0.5-40 ng of metalloprotease, the enzyme digested the substrate conjugates to release some of the biotin molecules so that the substrate conjugates have fewer or no biotin moieties. The released biotin molecules and the biotin moieties still attached to the substrate conjugates competed for the anti-biotin antibody in Capture Zone I to produce a weak blue signal, while some of the digested or partially digested substrate conjugates passed through Capture Zone I and were bound at Capture Zone II to produce a stronger blue signal.
When 40 ng of metalloprotease was used, all the biotin moieties were released from the substrate conjugates, and no more biotin was attached to the blue particles. The color intensity in Capture Zone I was very weak, and the blue color intensity in Capture Zone II was strong. In general, the color intensity of Capture Zone I decreased, and the color intensity of Capture Zone II increased as the amount of protease in the sample increased.
|Figure 6. Dose response curve for metalloprotease detection (Y axis: proportional reflectance, I2/ (I1+I2)).|
The color intensities of Capture Zones I and II can provide semi-quantitative or even quantitative measurements of the amount of active enzyme. The reflectance intensities of both Capture Zones I and II were measured using an in-house-developed scanner. Figure 6 shows the reflectance of Capture Zone II (I2) over the sum of Capture Zones I and II (I1 + I2) as a function of the amount of metalloprotease in a sample. The dose response curve shows a good linear correlation between the reflectance intensities and the amount of protease prior to saturation.
This article has demonstrated the adaptation of the lateral-flow format for detecting active enzymes. Lateral-flow enzyme assay techniques retain all the benefits of LFIA such as simplicity, user-friendliness, and low cost. Although this current study was focused on detecting the enzymes involving bond cleavage, the assay techniques could be adapted to enzymes involved in bond formation and conformational isomerization. In addition to detecting enzymes, the assay techniques should also be useful for detecting any molecular species involved in an enzyme reaction, such as an enzyme inhibitor. The technologies should find a wide variety of real-world applications such as POC and OTC tests for vaginal yeast infections (using spartyl proteases as biomarkers) and chronic wounds (using proteases from bacterial pathogens as biomarkers).
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Xuedong Song is a technical leader in corporate research and engineering at Kimberly Clark Worldwide (Dallas). He can be reached at firstname.lastname@example.org.