Quantum Dots as Förster Resonance Energy Transfer Acceptors of

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Quantum dots as Förster resonance energy transfer acceptors of lanthanides in time-resolved bioassays Sebastián A. Díaz, Guillermo Lasarte Aragones, Robert G Lowery, . Aniket, James N Vranish, William P Klein, Kimihiro Susumu, and Igor L. Medintz ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00613 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Quantum Dots as Förster Resonance Energy Transfer Acceptors of Lanthanides in Time-Resolved Bioassays Sebastián A. Díaz†,*, Guillermo Lasarte-Aragones†,Φ, Robert G. Lowery‡, Aniket‡,¥, James N. Vranish†,₤, William P. Klein†, Kimihiro Susumu§, Ω, Igor L. Medintz†,* †

Center for Bio/Molecular Science and Engineering, Code 6900, §Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, D.C. 20375, USA. Φ College of Science, George Mason University, Fairfax, Virginia 22030, United States ‡

Bellbrook Labs LLC, Madison, Wisconsin 53711 USA KeyW Corporation, Hanover, MD 21076 USA.

Ω

We report a flexible and modular design for biosensors based on exploiting semiconductor quantum dots (QDs) and their excellent Förster resonance energy transfer (FRET) acceptor properties along with the long-lived fluorescent lifetimes of lanthanide donors. We demonstrate the format’s wide application by developing a broad adenosine diphosphate (ADP) sensor with quantitative and high-throughput capabilities as a kinase/ATPase assay method. The sensor is based on a Terbium(Tb)-labeled antibody (Ab) that selectively recognizes ADP versus ATP. The Tb-labeled Ab (Ab-Tb) acts as a FRET donor to a QD which has an ADP modified His6-peptide conjugated to its surface via metal-affinity coordination. This strategy of using self-assembly modified peptides to present antibody epitopes on QD surfaces is readily transferable to other assays of interest. We utilize time resolved FRET (TRFRET) to measure the amounts of Ab-Tb bound to the QD by looking at the emission ratio of the QD and Tb in a time-gated manner, minimizing background signal. With the addition of free ADP the antibody is competitively separated from the QD and a change in the ratiometric emission signal correlates with the free ADP concentration. The sensor obtained a detection limit below 10 nM of free ADP and quantitation limit of 35 nM ADP using 8 nM of sensor. Quantitative values were obtained for a model enzyme (glucokinase) kinetics as well as demonstrations of the assays capability to distinguish enzyme inhibitors. We discuss future outlooks and note areas for improvement in similar design strategies. Keywords: quantum dots, lanthanides, time-resolved FRET, bioassay, ADP, enzyme kinetics.

Assays based on fluorescence are particularly suitable for high throughput screening as their simple readout can be optimized in microplate assays, allows for multiplexing at different wavelengths, and can be exploited through colorimetric and energy transfer methodologies. The toolbox of fluorescent molecules is in constant expansion beyond the traditional organic dyes; some of the recently exploited systems include inorganic nanoparticles such as semiconductor nanocrystals (quantum dots, QDs)1 as well as gold and silver nanoclusters.2, 3 QDs are particularly exploited for their bright, wavelength tunable emission.1 Another interesting option for fluorescent assays are chelated lanthanide ions, these have marked emission lines with large Stokes shifts, but what differentiates lanthanides from other emitters are their extremely long lifetimes which are in the µs-ms range.4, 5 This capability has found them exploited in a variety of different assays in what is often termed time-resolved Förster resonance energy transfer (TRFRET).4, 6 TR-FRET is different from fluorescence lifetime spectroscopy in that it does not concern itself on the arrival time of the detected photon, but includes a time-lag or timegate before the detection of the spectra or photon count.7 Time-gated detection in the µs range is advantageous for small molecule assays as it essentially eliminates background fluorescence from screening compounds, which typically have ns lifetimes. The most commonly used lanthanides for fluores-

cence are neodymium (Nd+3), europium (Eu+3), terbium (Tb+3), and ytterbium (Yb+3), yet it is important to remember that their absorption cross-sections are quite small and they require appropriate “antenna” molecules for adequate excitation.5 TR-FRET is generally exploited with organic dyes as acceptors, and in fact commercially available kits are available for a variety of targets based on this methodology (Bellbrook Labs, Perkin-Elmer, etc.). QDs may prove to be an improved acceptor option due to the large extinction coefficient, good quantum yields (QYs), and capability to act as center-symmetric scaffolds.1 In this manuscript we utilized Tb+3 as the lanthanide ion, from here on shortened to Tb, due to its emission range being in the visible and thus optimal for energy transfer to QDs.8 The recent work from multiple labs, detailed in a review from Dos Santos and Hildebrandt9, has shown that by utilizing a Tb-QD TR-FRET structure myriad applications such as spectroscopic rulers, immunoassays, and nucleic acid hybridization detection can be improved.6, 10-12 We believe that QDs as acceptors for lanthanides could be an excellent platform for TR-FRET bioassays, specifically for kinetic studies, due to the very large extinction coefficients, brightness, and sharp emission peaks of QDs allowing for sensitive and multiplexed detection. Our intention here was to develop a Tb-QD TR-FRET assay structure that is flexible, modular and with future applications in high-throughput bioassays. We report an

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initial evaluation of this design demonstrating its quantitative capabilities in an ADP assay, creating the first broad NP based TR-FRET assay for ADP that we could find in the literature, and note areas for future improvement. The adenosine phosphates, AMP, ADP, and ATP (adenosine mono, di, and tri-phosphate respectively), are key components in biocatalysis. Malfunctions in enzymes that produce ADP, such as kinases, ATPases, DNA helicases, etc., can have serious health consequences.13 Many of these enzymes are attractive drug targets and of great interest to both academics and industry.14 A few examples of QD based ADP assays are available, though ATP assays are much more common.15-18 The sensor is based on an ADP recognition agent, e.g. an antibody (Ab) or aptamer. Aptamers have binding constants in the µM range for ADP and ATP,19 but antibodies are available with nM affinity for ADP.20 A Tb-labeled Ab (Ab-Tb) acts as a FRET donor to a QD which has an ADP modified His6peptide ratiometrically conjugated to its surface via metalaffinity coordination.21 We utilize TR-FRET to measure the amount of Ab-Tb bound to the QD by looking at the emission ratio (ER) of the QD and Tb. In the presence of free ADP the antibody is competitively separated from the QD and a change in the ratiometric signal correlates with the ADP concentration (See Figure 1A for a schematic). Using this sensor design we obtained a detection limit below 10 nM of ADP (calculated using a signal-to-noise ratio of 3) and a quantitation limit of 35 nM ADP using 8 nM of sensor in a 20 µl of reaction volume.

EXPERIMENTAL SECTION: Synthesis of His6-ADP. 6-AB-ADP (N6-4-aminobutylADP, 2.5 µmoles. Biolog, Bremen, Germany) was combined with 3.5 µmoles GMBS (N-γ-maleimidobutyryloxysuccinimide ester, Thermo-Fisher) in 350 µl of 2× PBS (phosphate buffered saline) with trace DMSO for GMBS solubility. The reaction was allowed to proceed for 1 hour at RT at which point the peptide (Ac-HHHHHHSLGAAAGSGC, 3.1 µmoles) was added in 100 µl 2× PBS. The reaction continued at RT for 2 hours then was placed at 4 °C overnight. The ADP-peptide was purified using three 1 ml volume Ni-NTA columns using the dual-syringe method and then OPC cartridges.22 Fractions were dried down and then HPLC purified (see SI) to obtain the product His6-ADP (0.74 nmoles, Yield: ~30%). Sensor Formation. The active “Sensor” was formed by diluting CdSe/CdS/ZnS core/shell/shell QDs emitting at 605 ±10 nm with 8.2 ± 0.5 nm diameters coated with zwitterionic compact ligand CL4 (prepared as reported previously23, 24) into the reaction buffer (1.5× PBS + 5 mM Mg+2 + 0.05% Tween) to 200 nM (see SI for buffer selection details). This was combined with His6-ADP and dissolved in buffer in a 6 His6ADP/QD ratio. The peptide was allowed to bind for 60-90 min at RT after which the Ab-Tb was added and allowed to bind for > 30 min at RT. We note that the His6-tag has a binding constant to Zn+2 in the 1 nM21 range and therefore the amount of free peptide in our system should be negligible. Multiple Tb (from one to four) may actually be conjugated to a single Ab, yet as they act in conjunction as far as FRET “on” and FRET “off” is concerned, all the Tb on an Ab may be considered a single Tb with higher brightness. As only one Tb can act as the FRET donor per excitation, the D-A distance (rDA) can be considered as the average of all the Tb to the QD. Subsequent-

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ly we speak of Tb and Ab-Tb interchangeably when discussing donor-acceptor ratios. Spectral Detection. Fluorescence spectra were collected on a Tecan Infinite M1000 multifunction plate reader with timegating capabilities. Samples were prepared in 384 well Corning flat-black microtiter plates with 20 µl volume, with sensor concentrations at 8 nM based on QD concentration. For direct QD excitation without time gating, 340 nm excitation was used. Time-gated measurements were realized at 340 nm excitation with a time delay of 30-35 µs, with collection times varying from 300 to 500 µs, and 50 to 100 excitation flashes per measurement. All preparations were made in at least duplicate and control samples were run with each measurement series. Antibody Dissociation Constant. Spectral detection was the same as above. 50 nM of Ab-Tb was combined with varying concentrations of the commercially available reagent ADP-HiLyte647 (Bellbrook Labs, Madison, USA). The same experiment was realized with QD conjugated His6-ADP. Tb quenching and acceptor sensitization was detected and correlated to Ab binding. The data was then fit with the following equation:


= [] ⁄ + [] .

Eq. 1

Where [] is the concentration of either ADPHiLyte647 or His6-ADP, θ is the fraction of occupied binding sites, and KD is the antibody dissociation constant of interest.25 Displacement assays. The displacement assays were performed by measuring the spectra 20 min after addition of varying concentrations of free ADP to the Sensor solution. Control experiments with ATP were performed in a comparable manner replacing the free ADP with ATP. ADP and ATP were prepared fresh for each experiment from the corresponding salt by dissolving in buffer. Kinetic Assays of Enzyme Activity. The kinetic assays were carried out using 20 nM sensor and varying concentration of glucokinase (GLK) in the nM range. For full details on GLK please see Supporting Information (SI). Assays were prepared in 384 well Corning flat-black microtiter plates at 29 °C with 28 µl final volume and read on a Tecan Infinite M1000 multifunction plate reader with time-gating capabilities. Sensor and GLK was dispensed into wells and reactions were initiated by addition of substrate (final concentration 100 µM ATP and 120 µM glucose (glc)) immediately before emission detection. Control samples (Sensor and ADP) were premixed before addition to the plate and used to optimize detection conditions. The plate was shaken for 1 min and pre-heated to 29 °C before addition of substrate solution, shaken for another 10 seconds and then the intensity of the following three configurations was recorded every ~ 60 s: (1) QD Direct - 340 nm excitation, 605 nm detection, 0 µs of time gating, 50 flashes; (2) QD Time-gated - 340 nm excitation, 607 nm detection, 35 µs of time gating, 100 flashes; (3) Tb Time-gated - 340 nm excitation, 550 nm detection, 35 µs of time gating, 100 flashes. The plate was shaken for 5 s between each measurement. The kinetics were determined by observing the ratio of the QD and Tb signal in time gated modality. All measurements were realized in at least duplicate and each experiment was run with its own full set of controls. Data analysis was performed as detailed previously.26 Briefly: The progress curves were transformed into ADP production curves by utilizing the control values. The curves were converted into a progress curve as a

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ACS Applied Nano Materials function of time. By multiplying the reaction time (in seconds) by the enzyme concentration (in nM) for each measurement the data is converted into enzyme-time progress curves. Data were analyzed using the time-integrated version of the Michaelis–Menten (MM) equation:    

 ∗  =  +  ∗  

Eq. 2.

where kcat is the catalytic rate, E is GLK concentration, t is time, p is ADP concentration, KM is the Michaelis constant which reflects enzyme affinity for substrate, and a0 is the initial ATP concentration (100 µM).27 As detailed in previous work,26, 28 in general, obtaining the individual kcat and KM values is not possible due to requirements that broader ranges and excess substrate levels be tested. The ratio of the two parameters, kcat / KM, known as the specificity constant or catalytic efficiency can be obtained with confidence. This value is an effective second-order rate constant and of value when comparing a single enzyme with varying substrates or conditions.29 The methodology utilizes fits to the entire progress curves (not only the initial velocity) to determine the kinetic parameters, therefore experimental fluctuations should result in smaller uncertainty values. Inhibition Assays. The inhibitor screening assays were run in a very similar manner as the kinetics assays. The drug at a final concentration of 10 µM was added at the same time as the GLK enzyme (10 nM) with a wait time of 15 minutes before adding the substrate. Kinetic curves were obtained in a similar manner as above. The determination of the inhibitory constant for Palmityl-CoA (P-CoA) was realized in the same manner as the inhibitor screening assays, with varying concentration of P-CoA and 15 nM GLK. The required kinetic parameter was then obtained through the same analysis as detailed in the Kinetics section, with:  =  ∗ .30

RESULTS: Sensor Design and Characterization. The chosen strategy entailed exploiting the self-assembly of His6-tagged peptides on the surface of Zn+2 containing QDs as a means of presenting ADP to Ab-Tb.21 This required that we modify the peptide in a way that maintained the ADP epitope extended from the QD surface. Specific synthetic and purification details are available in the Experimental section and SI. 6-AB-ADP contains a readily available amine on a 5-bond linker that was modified with an amine-thiol crosslinker, which was then reacted with a short peptide containing a His6 section on the Nterminal (which was acylated to avoid undesired modification) as well as a single cysteine on the C-terminal (HHHHHHSLGAAAGSGC). The ADP-peptide was purified to obtain the product we refer to as His6-ADP. The His tag binds rapidly to Zn+2 ions on the QD surface through a self-assembly mechanism and in a ratiometric manner via metal affinity coordination.21 The rest of the peptide will extend from the QD surface and present any terminal moieties, making this an optimal conjugation strategy.31 We assessed several QDs, generally emitting in the 600-700 nm range and concluded that the key requirements were minimal emission overlap with the Tb emission peaks, high fluorescent QYs in aqueous conditions, smaller radius, and easily accessible surface area for conjugation. Accordingly we chose 605 ± 10 nm emitting CdSe/CdS/ZnS core/shell/shell QDs with a 4.1 ± 0.3 nm radius coated with CL4, a short zwitterionic ligand developed in house (see Figure 1).24 Efficient conjugation of the peptide to the QD was confirmed through agarose gel electrophoresis (see Figure 1C); migration is inversely related to the peptide/QD ratio. Binding of up to 50 peptides/QD was observed, though less were used in the final sensor design (typically 1 (where a value greater than one signifies positive cooperativity).30, 41 For small molecules, we tested Isatin (reported Ki = 9 µM) and Violuric Acid (Ki > 50 µM).46 In the case of the CoA molecules, palmityl-CoA (P-CoA, reported Ki = 2 µM) and acetyl-CoA (Ki > 10 mM) were evaluated.47 All tested molecules have a competitive inhibitory mechanism. Experiments were realized at minimum as duplicates with 10 µM of each drug. As can be seen in Figure 5 the P-CoA and Isatin have the strongest inhibitory effect with the Violuric acid showing none at all while Acetyl-CoA did not seem to modify the initial velocity of the reaction but had a lower completion percentage at the assay end point. The results mirrored expectations based on the reported Ki. Of interest is that the reaction endpoint (measured spectrally ~4 hours or 14000 s after substrate addition) of all the assays were indistinguishable within the uncertainty (see SI for spectra). This is consistent with the reversible nature of the competitive inhibition previously reported for the tested drugs.46, 47

Figure 4: Tb-QD TR-FRET kinetic assay of GLK enzyme. A) Schematic of GLK enzyme reaction. B) Generation of ADP plotted as a function of reaction time. Increasing GLK concentrations were utilized while all other conditions were kept constant. Additional control assays of the Sensor with no ATP nor ADP (noted as Sensor), a sensor system with excess ADP added previously (noted as ADP), and a reaction system in which the glc substrate was not added (No glc) were all tested and showed minimal ratio change. The fits are based on a MM mechanism,30 save for the controls in which the lines are guides for the eyes.

The slight rise effect seen for the “No glc” sample may arise from the excess ATP added to the reaction (~1000-fold excess) which, as seen in Figure 3B, does have a slight capability to displace the Ab-Tb resulting in a minor false positive. This hypothesis is supported by the lag of the observed curve as well as the 10% signal change which corresponds to the in vitro response observed with similar ATP excess previously. This data notes an important point, the assay must be optimized to initial concentrations of ATP. Though 5 orders of magnitude (10 nM to 100 µM) of ADP were tested successfully, greater amounts of initial ATP will commence to decrease the dynamic range of the assay. This can be solved by increasing the initial concentration of the sensor, always ensuring to keep the ATP ≤ 103 His6-ADP. Though the in vitro assays allowed for more than 60 min before the spectral analysis (to ensure comparability between experiments) the enzyme kinetics demonstrate that the equilibrium and read-out of the sensor is much quicker. The initial

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ACS Applied Nano Materials Figure 5: GLK inhibition assays. A) Kinetic assays of GLK enzyme (10 nM) with 10 µM of each drug. Curves are exponential best fits to the data. B) Determination of Ki for P-CoA at 16.6 nM GLK enzyme. A graph of the apparent enzyme kinetic parameter V/Km as a function of increasing concentration of inhibitor.

Further characterization was undertaken of the P-CoA in an attempt to determine its inhibitory constant (Ki). A similar experiment as above was undertaken with 0, 1, 5, 10, and 30 µM of P-CoA. As the mechanism of P-CoA was known to be competitive that meant that the following equation could be utilized to determine the Ki.30 

  ⁄ 

=

⁄!"

#$%'!

Eq. 3

&



Where   ⁄  were the experimentally determined kinetic parameter,  ⁄  was the value without inhibition, i was the inhibitor concentration, and Ki was the desired constant.30 The data and corresponding fit can be found in Figure 5B. The reported value in the literature was 2 µM47; experimentally we found a value of 1.0 ± 0.2 µM. Considering the different buffer conditions, as well as the difference from rat liver to E. coli GLK used in our experiments the proximity of the values is quite good.

CONCLUSIONS This proof-of-concept of a QD-Tb based TR-FRET bioassay, created a broad ADP sensor that is capable of working at the nM concentration for both sensor, analyte, and enzyme concentrations. Quantitative values were obtained for model enzyme kinetics as well as for inhibitory constants in line with those reported in the literature. The specific sensor was also capable of selectively discriminating enzyme inhibitor capabilities of structurally similar compounds. The sensor design may be exploited in a myriad of high-throughput assays in which a simple and economic detection method such as TR-FRET is used. This is advantageous in that many lanthanide based assays reported in the literature require a fluorescence lifetime detector,8 instrumentation of greater complexity and cost. When comparing the QD acceptor to the organic dye (HiLyte647) modified ADP analog we find two limitation in the QD system; 1) the size of the QD acceptor is not compensated by the increase in R0; 2) the longer lifetime and greater brightness of the QD results in higher background signal than the HiLyte647. As detailed the R0 for the Tb to QD was 7.6 nm, while the R0 for the Tb to HiLyte647 was 6.0 nm, yet the estimated rDA were 9.0 and 6.2 nm, respectively. So though the spectral overlap is smaller with the dye it is in much closer proximity to the Tb donor than the large QD (whose dipole is considered to originate at its center) can be. The QD also presents the limitation of having a much longer lifetime (20.0 ns)10 than organic dyes and higher brightness. This results in a background signal (see Figure 2) even with time-gating, which diminishes the ER change as compared to the HiLyte647. These issues can all be addressed, along with others that would improve the analytical capability of the probe. Smaller QDs, based on alloys, or even quantum rods with asymmetrical distribution could be exploited to decrease the rDA values.48, 49 Another route to minimize the rDA may be to use even shorter peptides and ADP linkers. Evidence that the current design did not cause decreases in the Ab KD values supports this pathway and we found that both acceptors were capable of working in similar sensor and analyte concentration ranges. Overall a

decrease of 1.2 nm in rDA is all that is required to put this QD based sensor on par with a commercial kit (Transcreener ADP² TR-FRET Red Assay, BellBrook Labs). We hypothesize that the increased R0 of QD acceptors as compared to organic dyes might be particularly beneficial when assaying larger analytes, where the relative rDA difference will be smaller than with ADP. Other mechanisms to increase the FRET may be obtained by improving QD chemistry: choosing one with greater spectral overlap with the Tb emission; lower absorption at 340 nm to minimize background signal, as well as further red-shifted QDs. The idea of exploiting QDs as acceptors for Ab-Tb donors could present other benefits such as further red-shifted emission and access to same-well multiplexing due to the QD’s sharp emission peaks.50 Similar assay designs for other targets such as ions, sugars, or modifications such as glycosylation are readily transferable.15, 51-53 The modular and multiplexing capabilities arise from the self-assembly of the modified peptides onto QDs of varying emission wavelengths. The chemistry of the peptide modification being the extensively utilized maleimide-NHS ester crosslinking strategy. The lanthanide donor could also be modified as recently reported Tbchelate structures have shown to be brighter and have greater QYs which would also increase the R0 values.54 Finally one could consider using other nanoparticles, for example gold nanoclusters can also emit in the far-red, have considerable spectral overlap with Tb emission, but are much smaller than QDs (~ 2 nm in diameter) and may even present enhanced energy transfer rates.2, 55, 56 In conclusion the use of QDs as scaffolds and FRET acceptors in TR-FRET bioassays should provide sensitivity in line with or beyond that currently available for organic dyes with an enhanced capability to multiplex in a single assay.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Buffer Selection, component ratio tests, HPLC purification of His6-ADP, Ab dissociation spectra, larger QD comparison, Streptavidin sensor spectra, competitive assay, GLK expression, Enzyme-Time curves, raw kinetics data, MM curves for GLK, inhibitor end-point kinetics spectra, P-CoA curves.

AUTHOR INFORMATION Corresponding Authors * [email protected], [email protected]

Present Addresses ₤ Ave Maria University, Ave Maria, FL 34142, USA. ¥ PerkinElmer Inc., Waltham, MA, 02451, USA.

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes Conflict of interest: BellBrook Labs manufactures and markets the Transcreener ADP² TR-FRET Red Assay used in part in this research. The authors, who collaborated in the research, declare no other potential conflicts of interest with respect to the research, authorship, and/or publication of this application note.

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ACKNOWLEDGMENT This work was supported by the Office of Naval Research (ONR) via the NRL Nanoscience Institute as well as by the Office of the Assistant Secretary of Defense for Research and Engineering (OSD R&E) via the Laboratory University Collaborative Initiative (LUCI) program. S.A.D. acknowledges a Karles Research Fellowship awarded by the NISE initiative.

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