Iridium(III) Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

The overall goal of this procedure is to detect the presence of a histidine rich malal protein biomarker in a sample using a phosphorescent iridium probe. This is accomplished by first synthesizing the cyclone methylated iridium complex. After a rapid chloride bridge splitting of the parent dimer complex, the cyclo methylated iridium complex can be isolated and purified by use in the assay.

The second step is to validate the interactions of the probe with various amino acids to ensure its specificity. For histamine alone, the phosphorescent signal is switched on only in the presence of histamine and is observed by luminescence.Spectroscopy. Next, as proof of concept, the iridium probe is titrated with a peptide mimic of the malaria biomarker, histidine rich protein two, to determine the limits of detection in solution.

Additionally, the kinetics of interaction of the probe with the peptide is elucidated using biolayer interferometry. The final step is to immobilize recombinant histidine rich protein on the surface of nickel NTA magnetic particles and assess the limit of the detection of the protein using the probe. Ultimately, lumins and spectroscopy is used to show how proteins can be detected on the surface of a solid support using a new class of phosphorescent iridium probes.

The main advantages of this technique over existing methods like fluorescent protein labeling and immunoassays, are that the probe is robust. It has fast association kinetics, and it's not susceptible to photobleaching. Additionally, this technique does not require lengthy time like immunoassays do.

The implications of this technique extend toward diagnosis of malaria infection? This is because of the high histidine content of the protein biomarker for plasmodium SRO infection called Histidine rich protein two. We first had the idea for this method when we noticed that a paper had been published using a cyclo methylated iridium probe to stain living cells.

In this paper, they stated that the probe entered the living cell and lit up the nuclei by binding to his seen rich proteins. Given the nature of the mal biomarker, his cine rich protein two, we decided to use this probe to detect our protein of interest. Visual demonstration of this method is critical due to the complexity of the assay and the numerous wash and transfer steps needed to detect this target protein on particle To begin way out, 53.6 milligrams of the parent iridium dimer and dissolve in five milliliters of methylene chloride.

Add this solution to a 50 milliliter Lummi flask equipped with a star bar. Then weigh out 26.2 milligrams of silver FL and dissolve in five milliliters of methanol. Add the dissolved silver FL to the parent iridium dimer solution and stir for one hour.

A cream colored slurry should result after one hour, filter the slurry through silica gel into a filter. Flask transfer the filtered product into a 25 by 95 millimeter DRAM vial evaporate off the remaining solvent using a rotary evaporator for five to 10 minutes. Once most of the solvent is removed, check that an oily yellow residue remains in the vial.

To this residue, add one milliliter of methanol to ol the product. Lay off ize for one to two days to yield a yellow solid and characterize the product as described in the text protocol to investigate the interactions of the iridium probe with various amino acids. Pipette 100 microliters of each amino acid solution into a black 96 well plate.

Add 100 microliters of he buffered saline or HBS to the plate to serve as a blank. Then add 2.5 microliters of the prepared two millimolar iridium probe solution to each sample and shake the plate on a plate shaker for 10 minutes. After 10 minutes, acquire the emission spectra of the samples using a 96 well plate reader.

To do so, place the plate in the instrument and open the plate reader software to set up a new experiment. Next, transfer the samples to a clear 96 Well plate and image the emission using a UV trans illuminator to perform titration of the iridium probe with the branched peptide mimic of the malaria biomarker, histamine rich protein two BNT two. First prepare a one milli molar stock solution of BNT two in HBS.

Then serially dilute the BN T two solution as described in the text protocol. Add 100 microliters of each dilution in triplicate with HBS serving as a blank to the wells of a 96 well plate to each sample, add 2.5 microliters of two millimolar iridium probe. Shake the plate for 10 minutes on a plate shaker after shaking, read the emission intensity at 510 nanometers using a 96 well plate reader.

Finally, transfer the samples to a clear 96 well plate and image of the titration using a UV trans illuminator to perform real-time kinetic analysis using biolayer interferometry. First, add 200 microliters of kinetic buffer to eight wells in the first column of a black 96 well plate. After inserting the plate into the plastic sensor holder, carefully transfer eight nickel a biosensors to the first column in the plastic holder such that the tips are suspended in the wells of buffer.

Then lease the plastic holder in the left side of the interferometry instrument to a new black 96 well plate pipette, 200 microliters of KB to all eight wells in columns one and three in the same plate, I bet 200 microliters of the 0.5 micromolar BNT two solution to the wells in column two. Then I bet 200 microliters of each dilution of the iridium probe into column four, starting with KB as the blank in well A four. Add the dilutions so that the wells increase in iridium probe concentration down the column.

Place this plate in the instrument to the right of the plate containing the pre wetting sensors in the biolayer interferometry software. Set up a basic kinetic experiment by defining the plate assay and sensor tips to define the plate, set a column on the screen right click and choose the appropriate definition. For the wells select buffer.

For columns one and three, load for column two and sample. For column four, define the assay in the next tab by first clicking add equilibrium. 60 seconds loading 120 seconds.

Association 120 seconds and dissociation 300 seconds. Select the equilibrium step and double click on column one. Then do the same for loading to column two, baseline to column three, association to column four and dissociation to column three.

Finally, select the nickel NTA sensors. Confirm the experiment is outlined correctly. Insert a file name.

Ensure that delay experiment is checked, and press go. Once the kinetic run is complete, process the data in the provided processing software to perform on bead detection of BNT two and recombinant HRP two with the iridium probe. Prepare serial dilution of BNT two and recombinant HRP two as described in the text protocol.

Then iPet 100 microliters of each dilution in triplicate into a white 96 well plate with the dilution in order from lowest to highest concentration down a column pipette 10 microliters of 50 micron nickel NTA magnetic arose particles into each dilution. Well make note that the magnetic particles remain suspended while pipetting. If they start to settle mixed by pipette or by vortexing, place the plate on a plate shaker for 15 minutes to incubate the samples with the particles after this incubation period, place the plate on a 96 well magnet and wait 30 seconds for the particles to pull out of solution.

Using a multi-channel pipette, pull off the original sample and discard as waste after removing the plate from the magnet at 200 microliters of HBS with tween or HBST to each well. Using the multi-channel pipette, pump the buffer up and down several times to wash the magnetic particles. Base the plate back onto the magnet and wait 30 seconds for the particles to pull out of solution.

Using the multichannel pipette, pull off the 200 microliters of buffer and discard as waste. Remove the plate from the magnet before repeating the washing steps two times to complete three washings of the particles. Then add 100 microliters of HBST to each well containing magnetic particles, followed by 2.5 microliters of the two millimolar iridium probe solution.

Incubate the particles with the iridium probe on a plate shaker for one hour. After one hour. Read the emission intensity at 510 nanometers using a 96 well plate reader.

After the synthesis and characterization of the cyclo methylated iridium probe, various amino acids are tested for reactivity with the complex. Only histidine elicits a signal response in relative fluorescence units upon titration of the probe. With two histamine containing peptides in solution, a concentration dependent response is observed.

This response can be seen here under broadband UV light. Biolayer interferometry for kinetic analysis of various concentrations of the iridium probe binding to BNT two on the surface of a nickel. NT.A glass sensor reveals that the iridium probe as micromolar affinity, the histidine containing peptides.

The limit of detection for the histidine rich protein using the probe was similar when the protein was immobilized on the surface of a magnetic particle. As when in solution, this labeling of the protein can be visualized on the surface of the particles themselves Once mastered. This technique can done in two hours if performed properly.

While attempting this procedure, it's important to remember to take care during the washing and transfer steps with the magnetic protein bound particles. Washing away unbound protein is important for proper detection of immobilized P-F-H-R-P two, Designing a molecular recognition element that will specifically target the bound protein through antibody antigen interactions. While still using the robust iridium probe will allow for more sensitive and specific detection of proteins bound on the surface of particles After its development.

This technique has the potential to be used to pave the way for researchers in the diagnostics field to explore utilizing such reagents that are robust for not only the detection of malaria, but for other infectious diseases.