Support for this work was provided by the Bill and Melinda Gates Foundation Grand Challenges in Global Health: Develop Technologies That Allow Assessment of Multiple Conditions and Pathogens at Point-of-Care. K.M.D. was supported by an NSF Graduate Research Fellowship (2012095464).

1. Synthesis of Iridium(III) Complex
<ol>
	<li>Weigh out 53.6 mg (50 &#181;mol) of [Ir(ppy)2-Cl]2 and dissolve in 5 ml of methylene chloride. Add this solution to a 50 ml Erlenmeyer flask equipped with a stir bar.</li>
	<li>Weigh out 26.2 mg (100 &#181;mol) of AgOTf and dissolve in 5 ml of methanol.</li>
	<li>Add the dissolved AgOTf to the [Ir(ppy)2-Cl]2 solution and stir for 1 hr. 
	Note: A cream colored slurry should result.</li>
	<li>After 1 hr, filter the slurry through silica gel into a 25 x 95 mm dram vial and evaporate off remaining solvent using a rotary evaporator (5-10 min).</li>
	<li>Once most of the solvent is removed, check that an oily yellow residue remains in the vial. To this residue, add 1 ml of methanol to re-dissolve the product and lyophilize 1-2 days to yield the yellow solid product.</li>
	<li>Characterize the Ir(ppy)2(H2O)2+ product ( Ir1) by H1NMR (in CDCl3), ESI and UV-Vis as described previously.23</li>
</ol>
2. Interactions of Ir1 with Various Amino Acids
<ol>
	<li>Prepare a 2 mM solution of Ir1 in methanol (MW 537.10 g/mol). Vortex to ensure complete dissolution of the solid.</li>
	<li>Prepare 100 &#181;M solutions of the following amino acids and biomolecules in HEPES buffered saline (HBS, 100 mM HEPES, 137 mM NaCl, pH 7.4) Ala, Asp, Cys, His, Ile, Lys, Ser, Try, Val.</li>
	<li>Pipette 100 &#181;l of each amino acid into a black 96-well plate. Add 100 &#181;l of HBS to the plate to serve as a blank.</li>
	<li>Add 2.5 &#181;l of the 2 mM Ir1 solution to each sample and shake the plate on a plate shaker for 10 min.</li>
	<li>After 10 min, acquire the emission spectra of the samples using a 96-well plate reader (365 ex/400-700 em).
	<ol>
		<li>Place the plate in the instrument and open the plate reader software to set up a new experiment.</li>
		<li>Set the new experiment to read a fluorescence scan with an excitation wavelength of 365 nm and a slit width of 9 nm with the optics at the top position.</li>
		<li>Read the emission from 300-700 nm with a step size of 5 nm.</li>
		<li>Export the data for data analysis.</li>
	</ol>
	</li>
	<li>Transfer the samples to a clear 96-well plate and image the emission using a UV Transilluminator.</li>
</ol>
3. Titration of Ir1 with BNT-II
<ol>
	<li>Prepare a 1 mM stock solution of BNT-II (MW 8233.6 g/mol) in HBS as well as a 2 mM solution of Ir1 in methanol.</li>
	<li>From the stock solution, prepare 1 ml of 100 &#181;M BNT-II in HBS. Vortex the microcentrifuge tube to ensure the sample is mixed.</li>
	<li>Serially dilute the 100 &#181;M BNT-II by half in HBS to a final concentration of 1.56 &#181;M BNT-II.</li>
	<li>Add 100 &#181;l 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 &#181;l of 2 mM Ir1.</li>
	<li>Shake the plate for 10 min on a plate shaker.</li>
	<li>After shaking, read the emission intensity at 510 nm (excitation at 365 nm) using a 96-well plate reader.
	<ol>
		<li>Place the plate in the instrument and open the plate reader software to set up a new experiment.</li>
		<li>Set the new experiment to read a fluorescence endpoint with an excitation wavelength of 365 nm and emission wavelength of 510 nm. Set the slit width for both wavelengths to 9nm, with the optics at the top position.</li>
		<li>Read the plate and export the data for analysis.</li>
	</ol>
	</li>
	<li>Transfer the samples to a clear 96-well plate and image the titration using a UV transilluminator.</li>
</ol>
4. Real-time Kinetic Analysis of the Ir1/BNT-II System Using Biolayer Interferometry
<ol>
	<li>Add 200 &#181;l of kinetic buffer (KB; 1x phosphate buffered saline with 0.02% Tween-20) to 8 wells in the first column of a black 96-well plate and insert the plate into the plastic sensor holder. Carefully transfer 8 Ni(II)NTA biosensors to the first column in the plastic holder such that the tips are suspended in the wells of buffer. Place the plastic holder in the left side of the interferometry instrument.</li>
	<li>Prepare a 2 ml of a 0.5 &#181;M solution of BNT-II in KB.</li>
	<li>Prepare 500 &#181;l of a 10 &#181;M solution of Ir1 in KB, and serially dilute by half down to 0.156 &#181;M in KB.</li>
	<li>To a new black 96 well plate, pipette 200 &#181;l of KB to all 8 wells in column A and C.</li>
	<li>In the same plate, pipette 200 &#181;l of the 0.5&#181;M BNT-II solution to the wells in column B.</li>
	<li>Pipette 200 &#181;l of each dilution of Ir1 into column D, starting with KB as the blank in well D1. Add the dilutions so that the wells increase in Ir1 concentration down the column.</li>
	<li>Place this plate in the instrument to the right of the plate containing the pre-wetting sensors.</li>
	<li>In the biolayer interferometry software, set up a basic kinetic experiment by defining the plate, assay, and sensor tips.
	<ol>
		<li>To define the plate, select a column on the screen, right click, and choose the appropriate definition for the wells. Select &#8220;Buffer&#8221; for columns A and C, &#8220;Load&#8221; for column B, and &#8220;Sample&#8221; for column D.</li>
		<li>Define the assay in the next tab by adding the following steps (Click &#8220;Add&#8221;): Equilibration (Custom), 60 sec; Loading, 120 sec; Baseline, 60 sec; Association, 120 sec; and Dissociation, 300 sec. Select the Equilibration step and double-click on column A. Repeat for Loading (column B), Baseline (column C), Association (column D), and Dissociation (column C). Select the Ni(II)NTA sensors.</li>
		<li>Move to the next tab and confirm that the sensors are in column A on the plastic sensor holder.</li>
		<li>Confirm the experiment is outlined correctly, insert a filename, ensure that &#8220;delay experiment&#8221; is checked, and press &#8220;Go.&#8221; 
		Note: Due to the automated nature of the instrument, once the kinetic experiment is outlined, the steps will occur as programmed.</li>
	</ol>
	</li>
	<li>Once the kinetic run is complete, process the data in the provided processing software.
	<ol>
		<li>Double-click on the folder with the filename of interest in the bottom portion of the panel on the left. Then, double-click the corresponding folder under &#8220;Kinetics&#8221; in the top portion of the panel. Move to the Processing tab.</li>
		<li>Check the Subtraction box on the left to open the sensor selection screen. Right click on well A4 and change the well type to Reference Well.</li>
		<li>Check the box next to Align Y axis, and ensure the selected step is Baseline. Specify the time range to be the last five sec of the baseline (approximately 55-60 sec).</li>
		<li>Check the box for Inter-step Correction and select Align to Dissociation. Ensure that the Savitzky-Golay Filtering box is checked and press &#8220;Process Data!&#8221;</li>
		<li>Move to the Analysis tab. Ensure that &#8220;Association and Dissociation&#8221; is the selection for steps to analyze and that the Model is 1:1. Select a Local, Full fit and press &#8220;Fit Curves!&#8221;</li>
		<li>Highlight all curves in the table and right click to set all curves to one color. Next, select a Global (Full) fit, grouped by color and Rmax Unlinked by Sensor. Press &#8220;Fit Curves!&#8221; to obtain global kinetics data.</li>
	</ol>
	</li>
</ol>
5. On-bead Detection of BNT-II and rcHRP-II with Ir1
<ol>
	<li>Prepare 1 ml each of a 1 &#181;M solution of BNT-II and rcHRP-II in HBS with Tween (HBST; HBS with 0.025% Tween 20).</li>
	<li>Serially dilute each protein by half in HBST to a final concentration of 15.6 nM.</li>
	<li>Pipette 100 &#181;l of each dilution, in triplicate, into a white 96-well plate, with the dilutions in order from lowest to highest concentration down a column.</li>
	<li>Pipette 10 &#181;l of 50 &#181;m Ni(II)NTA magnetic agarose particles into each dilution well.
	<ol>
		<li>Vortex the microcentrifuge tube containing the magnetic particles after each pipetting to ensure the particles stay suspended.</li>
	</ol>
	</li>
	<li>Place the plate on a plate shaker for 15 min to incubate the samples with the particles.</li>
	<li>After this incubation period, place the plate on a 96-well plate magnet and wait 30 sec for the particles to pull out of solution.</li>
	<li>Using a multichannel pipette, pull off the original sample and discard as waste. Remove the plate from the magnet.</li>
	<li>Add 200 &#181;l of HBST to each well using the multichannel pipette. Pump the buffer up and down several times to wash the magnetic particles.</li>
	<li>Place the plate back onto the magnet and wait 30 sec for the particles to pull out of solution.</li>
	<li>Using the multichannel pipette, pull off the 200 &#181;l of buffer and discard as waste. Remove the plate from the magnet.</li>
	<li>Repeat steps 5.8 through 5.10 two times to complete three washings of the particles.</li>
	<li>Add 100 &#181;l of HBST to each well containing magnetic particles followed by 2.5 &#181;l of 2 mM Ir1.</li>
	<li>Incubate the particles with Ir1 on a plate shaker for 1 hr.</li>
	<li>After 1 hr, read the emission intensity at 510 nm (excitation at 365 nm) using a 96-well plate reader.
	<ol>
		<li>Place the plate in the instrument and open the plate reader software to set up a new experiment.</li>
		<li>Set the new experiment to read a fluorescence endpoint with an excitation wavelength of 365 nm and emission wavelength of 510 nm. Set the slit width for both wavelengths to 9 nm, with the optics at the top position.</li>
		<li>Read the plate and export the data for analysis.</li>
	</ol>
	</li>
	<li>Transfer the samples to a clear 96-well plate and image the titration using a UV Transilluminator.</li>
</ol>

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The authors have nothing to disclose.

As microparticle-based diagnostic tools come to the forefront of modern diagnostic technology, detection of target biomolecules directly on the surface of the particle is a major advantage. Traditional molecular detection methods, such as ELISA and PCR, are advantageous, in that they can achieve very low limits of detection25. However, these assays require extensive reagents and lengthy protocols. This assay was designed to mimic ELISA-type sandwich interactions without the time and reagent requirements (Figure 5). Additionally, the cost of the Ir1 probe per sample was estimated to be around a penny, whereas the cost of antibodies for a typical ELISA is $0.20-0.30 per sample.
Since the methods described above rely on a cyclometalated iridium probe for detection of the malarial biomarker, this probe would not be susceptible to failure modes common to other detection methods (i.e. fluorophore quenching and antibody/enzyme degradation). Histidine is unique in its metal binding properties. The outlined work takes advantage of this phenomenon by replacing the antibodies in a typical ELISA sandwich with metal containing complexes. Ni(II)NTA captures rcHRP-II on the surface of the particle and Ir1 signals the presence of the protein. The most critical step in the assay is allowing the magnetic particles to mix with the sample and the probe. In a 96-well plate ELISA, the biomolecules and reagents reach equilibrium with the two-dimensional surface of the bottom of the well. Magnetic particles have the tendency to settle out of solution due to their dense iron-oxide core, which would reduce the available binding sites. Thus, the particles must be mixed during the assay time to ensure maximal binding.
When designing a reagent for disease diagnostics, one must keep in mind the form of patient sample as well as the physiological concentration of the biomarker. For malaria, the concentration of pfHRP-II in a patient&#8217;s blood can vary from low picomolar to high nanomolar. While this assay is clinically relevant for higher levels of infection, the limit of detection needs to be improved in order to detect asymptomatic patients with low picomolar circulating pfHRP-II. In the methods outlined above, the &#8220;switch-on&#8221; signal generated by Ir1 occurs in the presence of histidine. While the malarial biomarker is rich in histidine, other serum proteins, such as human serum albumin and histidine rich glycoprotein, would elicit a signal response with the probe. This would in turn lead to false positive diagnoses. This makes the capture of the protein on the surface of the particle a beneficial step, in that the protein of interest can be rapidly extracted from a patient sample. Additionally, designing a bifunctional probe, that couples a histidine rich peptide to a robust molecular recognition element (i.e. aptamer), could add another layer of specificity to the assay. The peptide would be loaded with iridium before coupling to the aptamer. In such a design, the stable Ir1 probe can still be utilized while achieving target specificity with the aptamer. This would allow for application of the probe to detect native HRP-II in a complex matrix (i.e. plasma or whole blood) and reduce non-specific binding effects. In order to further enhance the signal of rapid diagnostic tests, the assay could be incorporated into an electrochemiluminescent (ECL) system, where pfHRP-II can be detected on the RDTs as a electrochemical readout26. This next generation iridium probe would greatly enhance our on-bead ELISA assay for the detection of disease biomarkers.

Colorimetric and fluorescent labeling is an important method for the detection and tracking of biochemical molecules and processes1,2. The most common fluorescent markers are low molecular weight organic dyes3,4, but these molecules do not always have ideal optical properties. Fluorescent dyes are prone to photobleaching, often have small Stokes shifts, and may have overlapping excitation and emission spectra. Colorimetric labeling is often achieved by the use of enzymatic labels, which have an amplified signal useful in immunoassay quantification5,6. These enzymes also have their drawbacks, including photosensitivity, reaction conditions, and short substrate shelf life. These properties tend to require immunoassays and protein labeling methods to be done under well-controlled conditions using costly reagents.
Emissive transition-metal complexes have been explored as an alternative labeling approach for biochemical detection. In particular, cyclometalated Ir(III) has been studied in the context of organic light emitting diodes (OLEDs)7-9 oxygen sensing10, catalysis11, and protein/cell staining12-14. High photostability and quantum efficiency make this class of probes a good candidate for biomolecule detection15,16. It was previously found that cyclometalated Ir(III) complexes, of the form [Ir(C^N)2(solv)2]+, irreversibly bind histidine and elicit a blue-green signal response12,17. These complexes are non-emissive in the solvento state, but when histidine displaces the solvent molecules and binds to the metal center, they release an intense phosphorescent signal after long-wave UV irradiation. This signal only occurs after ligand substitution, and is the result of a triplet state electron relaxing to the ground state through the activation of metal ligand charge transfer (3MLCT) and ligand centered transfer (3LC) pathways8,15. These complexes can potentially be used as detection probes for histidine-rich proteins.
Histidine-rich proteins and their regulation levels are important in many diseases, including liver cirrhosis, cancer, and thrombic disorders.18Plasmodium falciparum Histidine Rich Protein II (pfHRP-II) in particular is a well-validated biomarker for malaria parasite infection. This protein is 67 kDa and contains 34% histidine, mostly within characteristic AHHAHHAAD repeating motifs19. These histidine repeats can bind free metal ions19 and heme complexes20 in host blood. pfHRP-II is commonly detected in low-resource settings using immunochromatographic rapid diagnostic tests (RDTs), but these tests are often inaccurate due to sample conditions, low biomarker concentration, poor manufacturing standards, and antibody degradation21.
Metal-based phosphorescent probes such as the cyclometalated Ir(III) complexes described above are attractive options for the detection of pfHRP-II due to their selective binding of histidine and their stabile and efficient emission properties. In this paper, the use of [Ir(ppy)2(H2O)2]+ (Ir1) to detect BNT-II, a branched peptide mimic of pfHRP-II is explored22. Kinetics of this interaction were monitored in real-time using biolayer interferometry techniques. The assay was also adapted to an on-bead ELISA-type format, where nanomolar limits of detection were achieved. This assay holds advantages over traditional ELISAs because it can be performed in under 2 hr with antibody-free reagents, instead of the 4-5 hr and biological reagents required with typical ELISAs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dicholortetrakis(2-(2-pyridinyl)phenyl)diiridium(III)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>658383</td>
			<td></td>
		</tr>
		<tr>
			<td>silver trifluoromethanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td>176435</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>Sigma-Aldrich</td>
			<td>6858</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Amino acids</td>
			<td>Fisher Scientific</td>
			<td>varies</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>BNT-II peptide</td>
			<td>Synthesized in house</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant HRPII</td>
			<td>Immunology Consultants Laboratory inc.</td>
			<td>AGPF-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar black polypropylene 96-well plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-567</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar white polypropylene 96-well plate</td>
			<td>Fisher Scientific</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
		<tr>
			<td>Grenier black polypropylene 96-well plate</td>
			<td>VWR</td>
			<td>89089-582</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(II)NTA magnetic agarose particles</td>
			<td>Qiagen</td>
			<td>36111</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(II)NTA biolayer interferometry sensors</td>
			<td>ForteBio</td>
			<td>18-5101</td>
			<td></td>
		</tr>
		<tr>
			<td>Octet Red96 Biolayer Interferometry Instrumet</td>
			<td>ForteBio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotek Synergy H4 Microplate Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher Biotech Transilluminator</td>
			<td>Fisher Scientific</td>
			<td>FBDLT-88</td>
			<td></td>
		</tr>
	</tbody>
</table>

iridium(iii) luminescent probe for detection of the malarial protein biomarker histidine rich protein-ii

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived phosphorescent signal when coordinated to a histidine-containing protein immobilized on the surface of a magnetic particle. Synthesis of Ir1, in high yields,is complete O/N&#160;and involves splitting of the parent cyclometalated Ir(III) chloro-bridged dimer into two equivalents of the solvated complex. To confirm specificity, several amino acids were probed for coordination activity when added to the synthesized probe, and only histidine elicited a signal response. Using BNT-II, a branched peptide mimic of the malarial biomarker Histidine Rich Protein II (pfHRP-II), the iridium probe was validated as a tool for HRP-II detection. Quenching effects were noted in the BNT-II/Ir1 titration when compared to L-Histidine/Ir1, but these were attributed to steric hindrance and triplet state quenching. Biolayer interferometry was used to determine real-time kinetics of interaction of Ir1 with BNT-II. Once the system was optimized, the limit of detection of rcHRP-II using the probe was found to be 12.8 nM in solution. When this protein was immobilized on the surface of a 50 &#181;m magnetic agarose particle, the limit of detection was 14.5 nM. The robust signal response of this inorganic probe, as well as its flexibility of use in solution or immobilized on a surface, can lend itself toward a variety of applications, from diagnostic use to imaging.

As shown in Figure 1, synthesis of the Ir1&#160;solvento-complex involved splitting of the chloride bridge in the parent dimer by precipitation of the chloride in the form of insoluble AgCl and association of water molecules to the metal center. The formation of Ir1 was confirmed by H1NMR and ESI. Additionally, UV-visible bands characteristic of metal ligand charge transfer and &#960;-&#960;* transitions were assigned to the spectrum, further validating the formation of Ir1. This solvated complex exhibits no emissive character when excited at 365 nm.
 
<img alt="Figure 1" src="/files/ftp_upload/52856/52856fig1.jpg" /> 
Figure 1: Synthesis and Characterization of Ir1. Chloride bridge splitting reaction of Dichlorotetrakis(2-(2-pyridinyl)phenyl)diiridium(III) to create the aquo complex Ir(ppy)2(H2O)2+ (Ir1). Upon addition of L-His to the aquo complex in aqueous buffer, luminescent signal is switched on.
Once the complex was synthesized and characterized, the amino acid selectivity was analyzed, as previously described using a similar iridium analog17. Figure 2A shows that only histidine elicited a signal response at 510 nm, when excited at 365 nm.
 
<img alt="Figure 2" src="/files/ftp_upload/52856/52856fig2.jpg" /> 
Figure 2: Amino Acid Selectivity and Titration of Ir1 with Histidine Rich Peptides. (A)&#160;Interaction of 200 &#181;M of various amino acids (Cys, Ser, Asp, Glu, Phe, Lys, Arg, Tyr, Trp, His) with 50 &#181;M Ir1 in HBS. Spectral scans were taken from 400-700 nm at an excitation wavelength of 365 nm in a black 96 well plate. Signal in relative fluorescence units (RFU) from all amino acids besides histidine (dashed black trace) was negligible. (B) Nanomolar concentrations of BNT-II (&#9642;) and rcHRP-II (&#9830;) were titrated with Ir1 in HBS. The histidine rich peptides were incubated with the probe for 15 min in solution before reading the emission at 510 nm after excitation with 365 nm light. Limit of detection was calculated as the value of x when y = 3&#963;blank.
This &#8220;on-switch&#8221; of the phosphorescence of the iridium probe occurs because the singlet excited state (1MLCT/1LC) of the Ir(III) bioconjugate undergoes intersystem crossing to the triplet excited state (3MLCT), when histidine is coordinated to the metal center. This probe was applied to BNT-II a peptide mimic of the malarial biomarker Plasmodium falciparum Histidine Rich Protein II (pfHRP-II). In a titration of BNT-II with Ir1, a concentration dependent signal response was observed (Figure 2B). Recombinant HRP-II (rcHRP-II) also exhibited a similar response in solution. The limits of detection of BNT-II and rcHRP-II in solution were 54.8 nM and 12.8 nM, respectively. Using biolayer interferometry techniques, the KD of Ir1 binding to BNT-II immobilized on a Ni(II)NTA surface was found to be 2.05 &#181;M (Figure 3).
<img alt="Figure 4" src="/files/ftp_upload/52856/52856fig4.jpg" /> 
 
Figure 3: Real-time Kinetic Analysis of Ir1 with BNT-II. Biolayer interferometry for kinetic analysis of various concentrations of Ir1 binding to BNT-II on the surface of a Ni(II)NTA glass sensor. After equilibrating the sensors in KB (Region A), the sensors are loaded with 0.5 &#181;M BNT-II (Region B). Once the peptide is loaded on the sensors, a baseline is established (Region C) prior to measuring the association of Ir1 to the BNT-II (Region D). After a period of association, the sensors are placed back into KB to measure dissociation (Region E). The whole kinetic analysis process takes less than 30 min.
When transitioning from a solution-based assay to a magnetic particle platform, the added complexity of the particle in the system had to be taken into account. It was known from previous work, that these particles efficiently bind the malaria biomarker pfHRP-II.24 Black fluorescent plates worked well for the solution assay describe above, but white plates were better suited for the on-particle detection platform, as seen in Figure 4A. In a white plate, the light is reflected back into the sample, thus allowing for better absorbance by the Ir1 bound to the surface of the particle. In the on-particle assay, the limit of detection of rcHRP-II was determined to be 14.5 nM (Figure 4B). The limit of detection for rcHRP-II in-solution and on-bead were statistically the same based on an unpaired t-test (p = 0.731).
<img alt="Figure 5" src="/files/ftp_upload/52856/52856fig5.jpg" style="font-size: 14px; line-height: 28px;" /> 
<img alt="Figure 5" src="/files/ftp_upload/52856/52856fig5.jpg" style="font-size: 14px; line-height: 28px;" />
 
Figure 4: On-bead Detection of BNT-II and HRP-II. (A) Difference between the signal detected from Ir1 bound to BNT-II on the surface of 50 &#181;m Ni(II)NTA magnetic agarose particles in a black 96-well plate (solid line) versus a white 96-well plate (dashed line). The particles were excited with 365 nm light, and emission was measured at 510 nm. (B) Titration of rcHRP-II immobilized on the magnetic particles and detected using Ir1. Limit of detection was calculated as the value of x when y = 3&#963;blank.
<img alt="Figure 5" src="/files/ftp_upload/52856/52856fig5.jpg" />
Figure 5: Schematic Representation of the On-bead Detection of rcHRP-II with Ir1. General scheme of HRP-II binding to the surface of Ni(II)NTA particles and labeled with Ir1. The particles are incubated with a histidine rich peptide for 15 min. After this incubation period, the particles are washed with HBST using a magnet, in order to pull the particles out of solution. Finally, the peptide bound particles are incubated with Ir1 for 1 hr prior to reading the emission at 510 nm after excitation with 365 nm light.

Watch this Scientific Journal Video about IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II at JoVE.com

IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

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

This work outlines the synthesis of a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which elicits a rapid, long-lived ...

iridiumiii-luminescent-probe-for-detection-malarial-protein-biomarker

Research

JoVE Journal

Chemistry

9.0K Views.  Vanderbilt University. Robust detection reagents are of increasing necessity for developing new malaria diagnostic tools. An iridium(III) probe was designed that emits long-lasting luminescent signal in the presence of a histidine-rich malarial protein biomarker. Detection of the protein either in solution or immobilized on a magnetic particle affords flexibility in application.

Video: IridiumIII Luminescent Probe for Detection of the Malarial Protein Biomarker Histidine Rich Protein-II

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Breath Collection from Children for Disease Biomarker Discovery

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, tuberculosis, lung cancer, and liver disease. This protocol describes a reproducible method for sampling breath in children and then stabilizing breath samples for further analysis with gas chromatography-mass spectrometry (GC-MS). The goal of this method is to establish a standardized protocol for the acquisition of breath samples for further chemical analysis, from children aged 4-15 years. First, breath is sampled using a cardboard mouthpiece attached to a 2-way valve, which is connected to a 3 L bag. Breath analytes are then transferred to a thermal desorption tube and stored at 4-5 &#176;C until analysis. This technique has been previously used to capture breath of children with malaria for successful breath biomarker identification. Subsequently, we have successfully applied this technique to additional pediatric cohorts. The advantage of this method is that it requires minimal cooperation on part of the patient (of particular value in pediatric populations), has a short collection period, does not require trained staff, and can be performed with portable equipment in resource-limited field settings.

This protocol describes a simple method for the acquisition of breath samples from children. Briefly, samples of mixed air are pre-concentrated in sorbent tubes prior to gas chromatography-mass spectrometry analysis. Breath biomarkers of infectious and non-infectious diseases can be identified using this breath collection method.

Breath collection and analysis can be used to discover volatile biomarkers in a number of infectious and non-infectious diseases, such as malaria, ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...