The overall goal of this study is to assess the diagnostic potential of direct and amplified surface plasmon resonance imaging, or SPRi assays, in the detection of biomarkers in serum, and compare it directly with the commercially available enzyme-linked immunosorbent assay kit. This method can help answer key questions in a biomedical field, such as highlighting the early stage of a disease, and providing means to monitor therapeutic efficacy. The main advantage of SPRi is that it allows label-free, real-time monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface.
The implications of this technique extend toward the diagnosis of hGH levels in serum, because endogenous hGH has been linked to numerous medical disorders that affect human growth and development. Visual demonstration of this method is critical, as the biochip surface chemistry preparation steps are difficult to learn, and any mistake can affect the results immensely. After preparing solutions and protein samples according to the text protocol, use 120 milliliters of stabilized piranha solution to clean the gold chip by sonication at 50 degrees Celsius for 90 minutes.
Use water to rinse the chip, and sonicate in water for five minutes. Then rinse the chip with ethanol and use a nitrogen stream to dry it. Place the gold chip in a UV ozone chamber for 30 minutes to remove any contaminants.
Next, add 150 milligrams of 11-mercaptoundecanoic acid to 20 milliliters of ethanol in a test tube containing a plastic cap to provide support for the chip, and a stir bar, to homogeneously mix the solution. Place the chip in the test tube and cap it. Then microwave the tube at 50 watts and 50 degrees Celsius for five minutes.
With ethanol, rinse the chip, and soak it in ethanol for five minutes. Use water to repeat the rinse, and soak for five minutes. Add 150 milligrams of 1-Ethyl-3-carbodiimide to 20 milliliters of water in a test tube containing a stir bar.
Transfer the chip into the test tube, and microwave as just demonstrated. Use water to rinse the chip, and soak it for five minutes in water. Then add 150 milligrams of N-Hydroxysuccinimide to 20 milliliters of water in a test tube with a stir bar.
Add the chip to the test tube, seal it with a cap, and microwave. After rinsing and soaking the chip in water as before, Add 250 micromolar polyethylene glycol in 20 milliliters of water to a test tube with a stir bar. Add the chip and microwave, then rinse and soak with water as before.
Prepare 15 micrograms per milliliter of anti-rhGH antibody solution, and anti-IgG as a negative control, and add 10 microliters of each to a separate well in a 384-well plate. Design the spotting pattern in the microarrayer, and with a 500-micrometer, teflon-tipped pin, spot the chip with the antibodies. Incubate the spotted chip at room temperature and 75%humidity for two hours.
After setting up the SPRi experiment, and blocking the chip according to the text protocol, prepare a solution of rhGH with the following set of concentrations in PBS, containing 10%serum, and one milligram per milliliter BSA. It is important to emphasize the type of blocking molecule chosen in this step. As the goal is to minimize the non-specific interactions.
Prepare a two-to-one solution by adding one microliter of six micromolar biotin-labeled anti-rhGH detection antibodies to three microliters of one micromolar streptavidin-coated near-infrared quantum dots in a 0.5 milliliter microcentrifuge tube. Inject 150 microliters of hGH-spiked human serum solution in the injection loop. A strong increase in signal will be followed by a slow drop from the non-specifically-bound serum rinsing off the surface.
Once the signal stabilizes, use a solution of 450 millimolar sodium chloride, added to the running buffer, to wash the surface. With running buffer, dilute the solution of anti-rhGH detection antibodies and quantum dots to a concentration of 10 nanomolar, and inject into the flow cell. Import the data into a data analysis program.
Then plot the SPRi signal versus time, and determine the difference between the anti-rhGH and negative control antibody spots after the high salt wash. To perform ELISA, after preparing the reagents, antibody-coated wells, and standards, according to the text protocol, prepare the samples of rhGH hormone in 10%human serum at the following concentrations. Add 50 microliters of each standard, control, and sample to each well in triplicate.
Then seal the wells, and incubate at four degrees Celsius with gentle shaking overnight. The following day, remove the plate from the shaker, and allow it to come to room temperature. Warm the anti-rhGH HRP, conjugate buffer, wash buffer, chromogen tetramethylbenzidine, or TMB, and stop reagent to room temperature.
To prepare the working reagents, use conjugate buffer to make a 40x dilution of anti-rhGH HRP. Use distilled water to dilute the wash buffer 200x. Add 50 microliters of anti-rhGH HRP conjugate into each well.
Seal the wells, and incubate at room temperature for 30 minutes with gentle shaking. Decant the solution from the wells, and invert the plate, and tap dry on absorbent tissue. Add 200 microliters of wash buffer into each well, before decanting, and tapping dry again.
Repeat the wash three times. It is important to tap the 96-well plate in order to remove accessory agents, as relying on the pipette is not effective. Next, add 100 microliters of chromogen into each well, within 15 minutes following the wash step.
Incubate at room temperature in the dark for 30 minutes with gentle shaking. The colorless solutions will turn blue. Add 100 microliters of the stop reagent into each well.
The solutions will turn from blue to yellow. Use a plate reader at 450 nanometers to immediately read the absorbance of each well. Finally, plot the standard curve for the provided standards, then plot the optical density of the rhGH in 10%serum samples, versus the concentration of each sample.
In this video, the performance of SPRi, and nano-SPRi, was compared with ELISA for the detection of rhGH. This figure represents the titration curve of rhGH-spiked in 10%human serum, and is plotted against the obtained OD at 450 nanometers. A good linear response was observed, and the limit of detection was determined to be one nanogram per milliliter.
Here, the detection of rhGH was assessed with SPRi. Each point on the concentration gradient curve represents the average value of the reflectivity difference calculated from three SPRi kinetic curves for each concentration. The limit of detection, or LoD, was determined to be 3.61 nanograms per milliliter.
To increase the sensitivity of the SPRi biosensor, nano-enhancers were sequentially introduced to the sensor. In this experiment, the nanoenhancers amplified the biosensor response up to 7.9%However, with minimal signal change to the control. The practicability of the nano-SPRi biosensor was assessed by measuring the range of rhGH from 30, 000 to 0.25 picograms per milliliter.
The LoD was calculated as 9.2 picograms per milliliter, and the coefficient of variation was 20%Once mastered, this technique can be done in one hour for direct detection, and two hours for nano-SPRi, if it is performed properly. While attempting this procedure, it is important to remember to optimize conditions for each step, and after doing so, results can be easily reproduced in a shorter duration. Following this procedure, other methods like multi-tuff, maspeck analysis can be performed directly on the chip after an SPRi experiment, in order to provide you with additional insights on the captured molecule.
After its development, this technique should pave the way for researchers in the field of medical diagnostics to explore detection of multiple biomarkers simultaneously, linked to cancer, neurological, and cardiovascular disorders. After watching this video, you should have a good understanding of how to set up a SPRi, a nano-SPRi, and ELISA assay for the detection of hGH levels in human serum. Don't forget that working with reagents can be extremely hazardous, and precautions such as wearing gloves and protective eyewear should always be taken when performing this procedure.
