Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody

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Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots Giacomo Annio, Travis Jennings, Oya Tagit, and Niko Hildebrandt Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00296 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Bioconjugate Chemistry

Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots Giacomo Annio,a,b Travis L. Jennings,c Oya Tagit,a,d,* and Niko Hildebrandta* a

NanoBioPhotonics (nanofret.com), Institute for Integrative Biology of the Cell (I2BC), Université ParisSaclay, Université Paris-Sud, CNRS, CEA, 91400 Orsay, France

b

Department of Medical Physics and Biomedical Engineering, University College London, London, UK c

d

Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA.

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands.

*Corresponding Authors Email: [email protected], [email protected]

Abstract: Quantum dots (QDs) are not only advantageous for colortuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient ABQD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QDbased immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance. Keywords: Biosensing, Fluorescence, Terbium, Time-gated spectroscopy, Nanoparticles 1 ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION

Colloidal semiconductor quantum dots (QDs) have been recognized as an attractive and versatile class of nanomaterials in optical biosensing due to their unique photophysical properties.1-4 The quantum confinement effect, arising from their very small dimensions, results in size-, composition-, and shape-tunable broad absorption spectra in the UV-visible-NIR range, and narrow emission peaks along with high photostability and brightness.5-7 Furthermore, their large surface areas and versatile surface chemistries enable immobilization of several recognition elements such as antibodies (AB) on QD surfaces via different conjugation strategies.8-13 Förster resonance energy transfer (FRET)-based immunoassays utilizing bioconjugated QDs represent a highly sensitive technique for biomolecule detection.14 FRET is a non-radiative process, in which energy is transferred from an excited donor (D) to an acceptor (A) in close proximity via dipoledipole coupling. In addition to molecular separation distances, the key parameters that influence the FRET efficiency are strongly related to the photophysical properties of D and A, such as the extent of spectral overlap between donor emission and acceptor absorption, relative orientation of their transition dipole moments, and quantum yield of the donor.15 In this context, QDs offer several advantages, in particular as FRET acceptors, due to their broad, continuous absorption bands, and high extinction coefficients.1,14,16 Luminescent lanthanide complexes (LLC) with multiple narrow and well-separated emission bands and extremely long excited-state lifetimes (a few milliseconds) are another interesting class of materials in FRET-based biosensing applications.17 When used as FRET donors, LLCs enable time-gated, background-free detection of biomolecules in homogeneous immunoassays, owing to their unusually long lifetimes. Furthermore, their well-separated emission peaks are ideal for simultaneous sensitization of several acceptors, which is necessary 2 ACS Paragon Plus Environment

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for multiplexed biosensing applications.18 In addition, unlike the common FRET pairs composed of conventional fluorophores, LLC-QD donor-acceptor pairs exhibit efficient energy transfer even at distances larger than 10 nm.19 Therefore, FRET pairs composed of LLC donors and QD acceptors can satisfy the requirements for rapid (homogeneous immunoassays without the requirement of washing and separation steps), sensitive (background-free) and multiplexed detection (several acceptors can be sensitized simultaneously) of biomolecules.20-22 Functionalization of QD surfaces with ABs is a prerequisite for the utilization of QDs in FRET-based biosensing. AB-QD conjugation approaches, however, may hamper the sensing performance if not designed carefully. The overall size of the AB-QD conjugates, correct orientation of AB on QD surface, and AB/QD conjugation ratio are among the several design criteria that can significantly influence the sensitivity of QD-based FRET immunoassays.23 The relatively large size of QDs is a cumulative result of the inorganic core, the organic coating for aqueous solubility, and the attached biomolecules3 and can be a challenge, particularly for sandwich immunoassays that require two relatively large AB conjugated to D and A. Size-related limitations have been alleviated in several studies via, e.g., conjugation of fragmented or singledomain AB instead of full-size AB,24-26 direct conjugation of AB to QD surface via their inherent thiol groups,27 or transfer of QDs to aqueous phase using zwitterionic small molecules and subsequent conjugation to AB using heterobifunctional linkers.28 Similarly, oriented conjugation of AB has been investigated to improve specificity, reactivity, and antigen binding capacity of AB for sensing applications.29-37 Site-directed conjugation of AB on QD surfaces has been achieved mainly through affinity interactions with a pre-formed layer of Fc binding proteins on QD surfaces,37 engineered monomeric single-domain AB,31,35 or via conjugating the fragmented AB using cross-linkers with flexible spacer arms.38

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Compared to AB size and orientation, it is more difficult to control the AB/QD conjugation ratios as the conjugation process inherently results in a heterogeneous distribution of the number of AB per conjugate, particularly when AB/QD ratios are kept below the QD surface saturation levels.39 Although fragmentation of AB has been shown to result in higher AB/QD conjugation ratios accompanied by an improvement of FRET-based detection sensitivity,24 it is difficult to relate the better sensitivity only to the presence of a higher number of AB fragments as fragmentation also results in smaller D/A separation distances. In this study, we use full-size AB (IgG) to decorate the QD surface at different AB/QD conjugation ratios and investigate the performance of each conjugate as FRET acceptors for the detection of total prostate specific antigen (TPSA) while leaving all the other assay components unchanged. After optical and colloidal characterization of AB-QD conjugates by various spectroscopic, microscopic, and chromatographic techniques, we quantify immunoassay performance regarding sensitivity, limit of detection, and dynamic range in time-gated FRET experiments, in which luminescent terbium complexes, Lumi4Tb (Tb), conjugated to a second AB with high affinity to TPSA (AB-Tb) act as donors.

RESULTS AND DISCUSSION Antibody-quantum dot conjugation Sulfhydryl-reactive eFluor 650NC QDs were supplied within an antibody conjugation kit composed of lyophilized QDs functionalized with activated maleimide groups, a conjugation buffer (PBS, pH 7.4), and a quencher (2-mercaptaethanol). The activated maleimide groups specifically target the thiol groups of AB, and the conjugation reaction is stopped by the addition of the quencher at the end of the incubation period. A detailed description of the conjugation chemistry has been reported elsewhere.40 In order to obtain different numbers of AB per QD, we 4 ACS Paragon Plus Environment

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used five different AB concentrations for conjugation to 20 µL of 3.3 µM activated QDs. Table 1 shows the initial molar concentrations of QDs and AB (and their ratios) used for each conjugation experiment. After conjugation, separation of unbound AB was performed through several centrifugation steps using 100 kDa spin columns. Although the molecular weights of IgG AB are in the range of 150 kDa, we previously showed that IgG AB can be adequately separated by this method.24,26 Absorption measurements and Bradford assays of flow-through liquids revealed that four washing cycles were sufficient to remove unbound AB (Fig. S1). Some of the flow-through liquids also contained QD PL signals (Fig. S2), which showed that small amounts of free QDs can penetrate the molecular-weight separation columns. Nevertheless, these free QDs were also completely removed after four washing cycles (Fig. S2).

Table 1. Concentrations and molar AB/QD reagent mixing ratios used in each conjugation experiment.

Sample #

0

1

2

3

4

5

0.066

0.066

0.066

0.066

0.066

0.066

AB (nmol)

0

0.06

0.12

0.23

0.58

0.92

AB/QD

0

0.9

1.8

3.5

8.7

14

Content QD (nmol)

Photophysical characterization of antibody-quantum dot conjugates Optical characterization was performed via absorption and PL emission and lifetime spectroscopy. After the last washing cycle, the concentrated samples were collected and the QD concentration was determined by UV-Vis spectroscopy. Then, samples were diluted to a final concentration of 18 nM for optical characterization. Absorption spectra of the conjugates revealed characteristic QD exciton peaks at 641 nm, 608 nm, and 515 nm (Figure 1a and 1b) and



a slight blue shift in the PL emission peak position compared to unconjugated QDs (Figure 1c). This hypsochromic shift in the band-gap emission has already been observed upon etching of the average nanocrystal size.41 In our experiments, the bioconjugation reaction was stopped by the addition of a thiol-containing quencher (2-mercaptoethanol), which targets the remaining active maleimide groups on the QD surface after AB conjugation. It is therefore not unlikely that these thiol groups reacted with the QD surface, which resulted in a certain degree of etching, manifested as a 2 nm hypsochromic shift in the PL emission spectra.

Figure 1. Optical characterization of unconjugated QDs (bright green) and AB-QD conjugates (18 nM) of varying conjugation ratios (black, red, blue, magenta and olive curves for Samples 1 to 5, respectively). Absorption spectra (a) (zoomed-in for the 500 nm – 700 nm range (b)), normalized PL emission spectra (c), and PL decay curves (d) of unconjugated QDs and AB-QD conjugates.


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PL decays (Figure 1d) were best described by a tri-exponential fit (Table 2). For all conjugates and unconjugated QDs, fast (~ 2.5 ns), moderate (~ 14 ns), and slow (~ 60 ns) components were observed. The amplitude-averaged lifetimes slightly increased with increasing conjugation ratios, which was evidence for improved surface stabilization by AB conjugation.

Table 2. Amplitudes and decay components of unconjugated QDs (0) and AB-QD conjugates of different conjugation ratios (Sample 1 to 5).

Sample #

A1

τ 1 (ns)

A2

τ2 (ns)

A3

τ 3 (ns)

(ns) * amplitudeaveraged

0

250

63.7

3300

13.6

7570

2.5

7.2±2.1

1

220

59.3

3710

13.9

7040

2.6

7.6±2.1

2

190

59.3

4040

13.8

6820

2.5

7.6±2.2

3

170

61.6

4330

14.1

6610

2.5

7.9±2.2

4

230

59.1

4690

14.8

6320

2.6

8.9±2.5

5

230

59.8

4680

15.2

6150

2.7

9.2±2.6

* = (A1τ1+A2τ2+A3τ3) / (A1+A2+A3). Errors were calculated via error propagation using 10% relative errors for the amplitudes (A1 to A3) and 20% relative errors for the decay times (τ1 to τ3).

Antibody-quantum dot conjugation ratios As a first approach to investigate AB/QD conjugation ratios, we used Bradford assays, which are frequently applied for protein quantification in analytical biochemistry.42 The increase of the absorbance at 595 nm is proportional to the amount of Coomassie Blue binding to proteins and therefore to the amount of protein present in the sample. Figure 2a shows the AB content in the AB-QD conjugates as a function of the initial amount of AB supplied for conjugation (cf. Table 1). Although the Bradford assays could clearly show a difference in the AB content between samples 1 to 3 and samples 4 and 5, the results also showed the weakness of this technique to



quantify low AB conjugation ratios, most probably caused by the strong absorption of the QDs at 595 nm. For the same reasons it was impossible to quantify low AB conjugation ratios by measuring the AB absorbance at 280 nm (results not shown), where the QD absorbance is even higher than at 595 nm. The Bradford results could also show that the conjugation efficiency is significantly below 100 %, in agreement with the absorption measurements obtained for the flowthrough liquids (Fig. S1a), in which, regardless of the initial concentration, a certain amount of free, unbound AB was detected. In a previous study, in which we used the same QDs, AB, and conjugation protocol but without variation of AB concentrations and with excess of AB during conjugation, we found a conjugation ratio of approximately 60 %.24 A theoretical 60 % conjugation efficiency curve (also shown in Figure 2a) is almost an adequate fit to the data points determined by Bradford assays, when taking into account a 30 % error. On the other hand, a constant conjugation ratio, independent of the initial AB/QD molar ratios, is not obvious. Low amounts of AB may slow down the conjugation process and a threshold concentration of AB may be required for efficient conjugation. High AB concentrations may lead to saturation of the QD surface or steric hindrance of AB competing for surface attachment. Such threshold at low concentrations and saturation at high concentrations would lead to a cubic curve, which would also be an adequate fit to the data in Figure 2a. Therefore, additional techniques are necessary to better evaluate AB-concentration-dependent QD conjugation. Because of the not entirely convincing results of the Bradford assays, we further investigated the AB/QD conjugation by Agarose gel electrophoresis and transmission electron microscopy (TEM). The photograph of the gel obtained under UV illumination (Figure 2b) clearly revealed a ‘retardation’ effect for AB-QD conjugates compared to unconjugated QDs. Due to the QD size distribution all samples displayed relatively broad bands. The migration distance relative to the loading well was determined for each conjugate and normalized to that of 8 ACS Paragon Plus Environment

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unconjugated QDs (Figure 2c). In contrast to Bradford, gel electrophoresis could clearly distinguish the lower conjugation ratios (Samples 1 to 3), which demonstrated the different amounts of AB on the QDs for these three conjugates. Similar to Bradford, a saturation became visible for the samples with higher AB/QD molar ratios (samples 4 and 5). Compared to unconjugated QDs, TEM of uranyl acetate stained AB-QD conjugates showed a brighter region around the QDs, which was more pronounced for higher AB/QD ratios (Fig. S3). Although only few AB-QDs were analyzed, these typical uranyl acetate staining regions provided another qualitative evidence for successful attachment of AB on the QD surfaces. FRET immunoassays (vide infra) also clearly showed a difference in the final conjugation ratios of all five samples.



Figure 2. (a) AB concentration of AB-QD conjugates as determined by Bradford assay versus the initial AB concentration supplied for conjugation experiments. The obtained data are within the range of a theoretical 60 % conjugation efficiency (red, dashed line). (b) Photograph of agarose gel obtained under UV illumination. Lane 1 (left): unconjugated QDs, Lanes 2-6: Samples 1 to 5, respectively. The red arrow shows the migration direction. (c) Relative migration distance of AB-QD conjugates normalized to the migration distance of unconjugated QDs.


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Tb-to-QD FRET immunoassays The performance of QD-based FRET immunoassays can be expected to depend on the valency of AB/QD conjugation. Multivalency may have positive effects when it comes to binding of multiple targets to one single AB-QD conjugate but it may also have negative effects when target binding is blocked due to steric hindrance on the functionalized QD surface. Valencies close to or below unity mean that the assays contain QDs with and without AB. QDs without AB may significantly contribute to background signals without performing any biological binding function. Background effects of QDs without AB may be reduced by washing or the use of the QD-conjugates as FRET acceptors in combination with long-lifetime FRET donors. Another important aspect to consider is the cost of the bioconjugates because primary antibodies can be quite expensive and an economic use may be desired. All these considerations show that it is difficult to predict the exact effect of AB-QD conjugation on assay performance. To better understand the influence of AB-valency on QD surfaces, we used Tb-to-QD FRET immunoassays against TPSA, a homogeneous immunoassay system we had previously studied with a constant AB conjugation ratio.24 In these immunoassays, a Tb-conjugated AB and the AB-QD conjugate both bind to the target PSA (formation of a so-called sandwich complex), which leads to FRET from Tb to QD and time-gated intensities of both FRET-quenched Tb and FRET-sensitized QD PL are used for TPSA quantification. Optical characterization of Tb-AB complexes is shown in Fig. S4. The molar absorptivity spectrum of conjugates displayed two distinct peaks around 340 nm and 280 nm, which corresponded to absorption of Tb and AB, respectively (Fig. S4a). Based on the absorption spectrum, Tb/AB conjugation ratio was estimated to ~14 Tb per AB. The Tb-AB conjugates displayed well-separated emission peaks of Tb3+ in the 480 to 700 nm spectral range (Figure 3a) and an almost single-exponential decay with an amplitude-averaged PL lifetime of 2.61 ms (Fig. S4b). The overlap integral for Tb-AB 11 ACS Paragon Plus Environment


emission and AB-QD absorption (Figure 3a) was J = 1.52x1017 nm4M−1cm-1, which led to a Förster distance of R0 = 10.6 nm (see SI for details). Tb-donor and QD-acceptor PL decays (Figure 3c) were measured in parallel in two detection channels using two band-pass filters at 495±10 nm (donor channel: ChD) and 660±6.5 nm (acceptor channel: ChA). A time-gated intensity ratio (FRET Ratio) was calculated from the ChA and ChD PL intensities in a timewindow ranging from 0.1 ms to 0.9 ms.

Figure 3. (a) Spectral overlap of Tb-AB PL (green) and AB-QD molar absorptivity (red). (b) PL spectra of Tb-AB (green) and AB-QD (red) and transmission spectra (blue) of the bandpass filters used for the detection of Tb PL (ChD) and QD PL (ChA). (c) PL decay curves obtained in ChD (left) and in ChA (right) for varying TPSA concentrations for sample 5. The sensitized emission in ChA increased with increasing TPSA concentrations from 0 nM (black) to 18 nM (green). Purple: 1.5 nM, dark green: 3.0 nM, blue: 4.5 nM TPSA.


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For all FRET experiments the concentrations of Tb ([Tb] = 42 nM; [AB] = 3 nM) and QD ([QD] = 0.25 nM; variable AB concentrations depending on the AB-QD conjugate) were kept constant and FRET ratios were measured for varying TPSA concentrations at different incubation times. A representative set of decay curves obtained in ChD and ChA for sample 5 is shown in Figure 3c. The FRET-sensitized PL intensity in ChA increased with increasing TPSA concentrations, whereas the PL intensity in ChD was almost unchanged, due to the many Tb (14 per AB) that did not transfer their energy to QD acceptors. Although different incubation times (from 45 min to 3h) did not significantly change the results, 135 min was found to be optimal for all samples (Fig. S 5). The TPSA assay calibration curves for all five different AB-QD conjugates (Figure 4) increased with increasing TPSA concentrations until they reached a signal saturation and leveled off into a plateau region as commonly found for such non-competitive binding assays.24 The significant differences in the assays also confirmed the variations in AB/QD conjugation ratios. Figure 4a revealed strong differences in both the steepness of the curves as well as their maximum FRET Ratio values that were reached at saturation. Both increased with increasing AB/QD conjugation ratios. A closer look at the single curves (Figures 4b to f) showed that all assays were functional (typical assay calibration curves) and that both the degree of maximum FRET sensitization (numbers indicated in blue) and TPSA saturation concentrations (numbers indicated in red) were considerably higher for samples with higher AB/QD ratios. In a previous study about FRET assays based on biotin-streptavidin binding, we used the saturation concentrations to determine the number of biotins on the QD surface.43 In principle, the same approach should also be applicable to determine the number of AB per QD in our FRET-based immunoassays because the Tb-AB were in excess (3 nM) and therefore saturation should have occurred when no more AB-QD were available. Dividing the saturation concentrations (1.3, 1.7, 13 ACS Paragon Plus Environment


1.6, 2.1, and 3.0 nM) by the QD concentration (0.25 nM) would lead to 5.2, 6.8, 6.4, 8.4, and 12.0 AB per QD for the five different samples. In comparison to the initial molar ratios that were used for AB-QD conjugation (0.9, 1.8, 3.5, 8.7, and 14 – cf. Table 1) the AB/QD ratios determined from the saturation concentration are significantly too high, in particular for the first three samples. These unsatisfying results can be explained by the FRET-immunoassay itself. In contrast to biotin-streptavidin assays, FRET-immunoassays have two binding events with three binding partners (two AB and TPSA) and the dissociation constants are significantly higher (below pM for biotin-streptavidin and in the nM range for AB-antigen) and were in the same range as our saturation concentrations. Moreover, the saturation concentration is the one at which all binding sites are saturated. The monoclonal IgG AB used in our study have two identical antigen binding sites each. However, when these IgG are attached to a QD, the availability of the binding sites also depends on the orientation and on steric hindrance. This situation gets even more complicated because the second AB (Tb-AB) also needs to bind the antigen and again with two identical binding sites. Finally, the maximum FRET sensitizations were quite small for samples 1 to 3 (increases of 4.5%, 17%, and 38%, respectively), which made the accuracy even lower for these samples. As both the degree of maximum FRET sensitization and the TPSA saturation concentration were strongly influenced by the AB valencies, we decided to use their product (assay saturation product = maximum FRET sensitization x TPSA saturation concentration) to evaluate the AB/QD conjugation ratios.


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Figure 4. Combined (black, red, blue, magenta, and olive for Samples 1 to 5, respectively) (a) and individual (b to f) TPSA assay calibration curves (FRET Ratio as a function of TPSA concentration normalized to unity at zero TPSA concentration). Error bars for [TPSA] = 0 represent the standard deviation from five individual measurements. For all other concentrations, error bars represent the standard deviation from three individual measurements. Grey and blue lines represent the linear increasing and saturation portions of the curves, respectively. The inflection points (intersection point of both lines) are shown in red and corresponding FRET Ratio values at saturation are depicted in blue.

In contrast to the evaluation with Bradford assays, Agarose gel electrophoresis, or the saturation concentration alone, the assay saturation product showed a linear increase with increasing molar AB/QD ratio (Figure 5). Although none of the characterization techniques was able to quantify the exact number of AB per QD, the best and most efficient relative evaluation of AB-QD conjugation was accomplished by the FRET immunoassays. The method is not limited by low or high AB-conjugation ratios, no additional characterization technique is required, and the evaluation can be directly performed under the actual assay conditions at very low reagent concentrations. Nevertheless, each of the characterization techniques has its specific advantages



and limitations (Table 3) that must be taken into account and evaluated in view of the specific conditions and requirements of the AB-QD conjugate application. 8

assay saturation product

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7 6 5 4 3 2 1 0

2

4

6

8

10

12

14

molar AB/QD ratio Figure 5. Assay saturation product (product of the blue and red assay parameters from Figures 4b to f) as a function of the initial molar AB/QD ratios used for conjugation (cf. Table 1). The red line is a linear fit through the data points. Error bars present maximum errors as calculated from the assay parameter errors – cf. Figure 4).

Table 3: Advantages and limitations of AB-QD conjugate characterization methods

Characterization Technique

Advantages

Disadvantages

UV-Vis spectroscopy

simple; in solution

requires relatively high concentrations; extremely high absorbance of QDs in the UV

Bradford assay

commercially available; in solution

requires additional dye; requires relatively high concentrations; high absorbance of QDs in the Vis

Agarose gel

good visual distinction (fluorescence) by size

requires relatively high concentrations; broad bands; only qualitative

TEM

direct visualization with nanometer resolution

dried sample on grid; requires protein staining; complicated/large equipment; only qualitative

FRET-assay

low concentrations; in solution; actual assay conditions

difficult quantification due to multiple binding sites and sandwich binding type


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Finally, we tested the dependence of the sensing performance of the TPSA immunoassay on the number of AB per QD by comparing the limits of detection (LOD) and dynamic ranges using the five different AB-QD conjugates (Table 4). LOD is the minimum detectable sample concentration4 and was determined (from the calibration curves in Figure 4) as the concentration that corresponded to the FRET Ratio of three times the standard deviation above the blank (no target). A large dynamic range is important to cover a wide range of target concentrations in diagnostic assays and was defined as the concentration difference between LOD and saturation concentration. Increasing the initial AB/QD ratio from 0.9 to 14 led to an 8-fold improvement of the LOD (703 pM to 85 pM) and a 5-fold improvement of the dynamic range (0.6 nM to 3.0 nM), which showed the strong influence of AB/QD conjugation ratios on assay performance. The importance of evaluating the assay becomes clear when taking into account the clinical cut-off value of PSA (4 ng/mL).44 In this regard, only conjugate samples 4 and 5 could provide sufficiently low LODs.

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

LOD (pM)

703

243

150

94

85

LOD (ng/mL)

23

7.8

4.8

3.0

2.7

Dynamic range (nM)

0.6

1.5

1.5

2.0

3.0

Table 4. LOD and dynamic range of immunoassays determined by time-gated FRET analysis.



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CONCLUSION Controlled conjugation of QDs with AB is highly important for their utilization in FRET-based biosensing applications. In this work, we demonstrated the importance of the AB valency (number) in AB-QD conjugates for their biosensing performance by applying different AB/QD conjugation ratios to Tb-to-QD FRET immunoassays against TPSA. Although absorption and emission spectroscopy, Bradford assays, Agarose gel electrophoresis, and TEM could provide useful information regarding the actual conjugation of AB to QDs with different initial molar ratios of AB and QDs, the analysis of the immunoassays itself (saturation concentration and maximum FRET sensitization) resulted in the best evaluation under actual assay conditions and showed a clear linear dependence of AB-QD conjugation on the initial molar AB to QD ratios. An eight-fold improvement of the LOD and a five-fold improvement of the dynamic range was accomplished by increasing the initial molar ratio from 0.9 to 14. Our findings show the importance of a careful characterization and optimization of AB valencies in AB-QD conjugates for the development of efficient and sensitive FRET immunoassays or other AB-based biosensors.

MATERIALS AND METHODS Reagents.

Sulfhydryl-reactive

CdSe/ZnS

core/shell

QDs

with

malaimide

surface

functionalization (eFluor®NC650) and QD/antibody conjugation kit (eFluor® Nanocrystal Conjugation Kit Sulfhydryl-Reactive) were obtained from eBioscience (San Diego, USA). The NHS-activated terbium complex Lumi4-Tb in lyophilized form was generously provided by Lumiphore, Inc. (Berkeley, USA). Prostate specific antigen (PSA) and monoclonal primary 18 ACS Paragon Plus Environment

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antibodies against PSA (IgGs “PSR222” and “PSS233”) were provided by Cezanne/Thermo Fisher Scientific (Nîmes, France). Agarose powder, bovine serum albumin (BSA) and all ingredients for buffer preparation (PBS, Tris(hydroxymethyl)-aminomethane (TRIS/Cl) and sodium tetraborate, carbonate) were purchased from Sigma-Aldrich. Preparation of AB-QD conjugates. AB-QD conjugates of varying AB content were prepared using the conjugation kit supplied by eBioscience in five different AB/QD ratios. Briefly, lyophilized QDs were wetted with 100 µL of conjugation buffer and the vial was placed into a water bath at 60°C with gentle flicking until an optically-clear solution was obtained. The QD solution was split within five separate vials (20 µL each) and mixed with 5 µL (Sample 1), 10 µL (Sample 2), 20 µL (Sample 3), 50 µL (Sample 4), and 80 µL (Sample 5) of 1.73 mg/mL PSS233 (IgG antibody) solution. The mixtures were gently agitated at 30 rpm using an ELMI Intelli-Mixer shaker at room temperature for 2 h. The reaction was stopped by adding a quencher (2-mercaptaethanol) supplied with the kit and the conjugates were purified by centrifugation at 5000 rpm for 20 minutes, (Eppendorf 5424R centrifuge) using 100 kDa spin columns (Millipore). The samples were washed four times using sodium tetraborate buffer (100 mM, pH 8.3) and flow-through liquids were collected after each cycle for spectroscopic analysis. Preparation of Tb-AB conjugates. Lumi4-Tb-NHS was reacted in molar excess to available primary amines of the PSR222 IgG antibodies by mixing both solutions in carbonate buffer (pH 9). The mixtures were incubated for 2 h at room temperature while rotating at 30 rpm using an ELMI Intelli-Mixer shaker. The Tb-AB conjugates were purified and washed four times with 100 mM TRIS/Cl pH 7.4 using 50 kDa molecular weight cut-off spin columns (Millipore) and a Heraeus MegaFuge (Thermo Scientific). Optical characterization. QD concentrations were determined by absorbance measurements on a Perkin Elmer Lambda 35 UV/vis system using a molar absorptivity of 1.1 × 106 M–1 cm–1 as 19 ACS Paragon Plus Environment


provided by the manufacturer. Emission spectra and photoluminescence (PL) decay times were recorded on a PicoQuant FluoTime 300 spectrometer equipped with a diode laser (centered at 405 nm, operating at 2 MHz, Edinburgh Instruments) or a Xe-flash lamp operating at 100 Hz for AB-QD and Tb-AB conjugates, respectively. Protein quantification of AB-QD conjugates was performed using a commercial Bradford assay (Bio-Rad Protein Assay). Colloidal characterization. Agarose gel electrophoresis was performed on a 0.5 X agarose gel prepared with tris-borate-EDTA buffer. 10 µl of 50 nM sample solutions were run at 100 V for 30 minutes and the gel was imaged using a Gel Doc™ EZ UV gel scanner (Bio-Rad). Transmission electron microscopy (TEM) was performed on a Jeol 1400 TEM (120 kV) with uranyl acetate staining. A drop of 5 nM AB-QD conjugate solution was placed on top of a carbon-coated copper grid and imaged after uranyl acetate staining. Time-gated FRET experiments. Förster distances and spectral overlap integral were calculated as described elsewhere.44 Time-gated FRET-based immunoassays were performed on a custom-made fluorescence plate reader (Edinburgh Instruments) using a 96-well 150 µl microtiter plate. A nitrogen laser VSL 337 ND (Spectra Physics) (337 nm) operating at 20 Hz (~120 µJ pulse energy) was used as the excitation source. The plate reader used 4000 detection bins of 2 µs integration time and detected donor and acceptor PL by two photomultiplier tubes (PMT). A dichroic mirror (500 nm, Semrock) was used to split the emitted PL to the PMTs with bandpass detection filters (Semrock) for donor (495/20 nm) and acceptor (660/13 nm) signals. Tb-AB donor and AB-QD acceptor conjugates were mixed in fixed assay concentrations of 3 nM (based on AB concentration) and 0.25 nM (based on QD concentration), respectively, and incubated for 135 min after addition of TPSA in gradually-increasing concentrations. FRET ratios were determined for each TPSA concentration.


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Acknowledgments. The authors thank Lumiphore, Inc. for the gift of Lumi4 reagents and the European Commission (FP7, grant agreement n°246556) for financial support.

Supporting Information. Absorbance measurements at 280 nm, Bradford assays (both Figure S1) and emission spectra (Figure S2) of flow-through solutions measured after each washing step; TEM images of AB-QD conjugates (Figure S3); optical characterization of Tb-AB conjugates (Figure S4); influence of incubation time on the FRET ratios for each conjugate (Figure S5); and calculation of the FRET parameters. This Supporting Information is available free of charge on the ACS Publications website.

REFERENCES (1) Geißler, D. and Hildebrandt, N. (2016) Recent developments in Förster resonance energy transfer (FRET) diagnostics using quantum dots. Anal. Bioanal. Chem. 408, 4475-4483. (2) Martynenko, I. V., Litvin, A. P., Purcell-Milton, F., Baranov, A. V., Fedorov, A. V., and Gun'ko, Y. K. (2017) Application of semiconductor quantum dots in bioimaging and biosensing. J. Mater. Chem. B 5, 6701-6727. (3) Sapsford, K., Pons, T., Medintz, I., and Mattoussi, H. (2006) Biosensing with Luminescent Semiconductor Quantum Dots. Sensors 6, 925-953. (4) Tagit, O. and Hildebrandt, N. (2017) Fluorescence Sensing of Circulating Diagnostic Biomarkers Using Molecular Probes and Nanoparticles. ACS Sensors 2, 31-45. (5) Alivisatos, A. P. (1996) Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science 271, 933-937. (6) Bao, J. and Bawendi, M. G. (2015) A colloidal quantum dot spectrometer. Nature 523, 67-70. (7) Tagit, O., Annio, G., and Hildebrandt, N. (2015) Terbium to quantum rod Förster resonance energy transfer for homogeneous bioassays with picomolar detection limits. Microchim. Acta 182, 1693-1700. (8) Clapp, A. R., Goldman, E. R., and Mattoussi, H. (2006) Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins. Nat. Protocols 1, 1258-1266. (9) East, D. A., Todd, M., and Bruce, I. J. (2014) Quantum Dot–Antibody Conjugates via Carbodiimide-Mediated Coupling for Cellular Imaging. Quantum Dots: Applications in Biology (Fontes, A., Santos, B. S., Eds.) pp 67-83, Springer, New York. (10) Han, H.-S., Niemeyer, E., Huang, Y., Kamoun, W. S., Martin, J. D., Bhaumik, J., Chen, Y., Roberge, S., Cui, J., Martin, M. R. et al. (2015) Quantum dot/antibody conjugates for in vivo cytometric imaging in mice. Proc. Natl. Acad. Sci. U.S.A. 112, 1350-1355. (11) Hines, D. A. and Kamat, P. V. (2014) Recent Advances in Quantum Dot Surface Chemistry. ACS Appl. Mater. Interfaces 6, 3041-3057. (12) Kilina, S. V., Tamukong, P. K., and Kilin, D. S. (2016) Surface Chemistry of Semiconducting Quantum Dots: Theoretical Perspectives. Acc. Chem. Res. 49, 2127-2135.



(13) Zhou, J., Yang, Y., and Zhang, C.-Y. (2015) Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application. Chem. Rev. 115, 11669-11717. (14) Hildebrandt, N., Spillmann, C. M., Algar, W. R., Pons, T., Stewart, M. H., Oh, E., Susumu, K., Díaz, S. A., Delehanty, J. B., and Medintz, I. L. (2017) Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications. Chem. Rev. 117, 536-711. (15) van der Meer, B. W. (2013) Förster Theory. FRET – Förster Resonance Energy Transfer (Medintz, I.L. and Hildebrandt, N., Eds.) pp 23-62, Wiley-VCH Verlag GmbH & Co. KGaA. (16) Chou, K. and Dennis, A. (2015) Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors. Sensors 15, 13288. (17) Hildebrandt, N., Wegner, K. D., and Algar, W. R. (2014) Luminescent terbium complexes: Superior Förster resonance energy transfer donors for flexible and sensitive multiplexed biosensing. Coord. Chem. Rev. 273–274, 125-138. (18) Zwier, J. M. and Hildebrandt, N. (2016) Time-Gated FRET Detection for Multiplexed Biosensing. Reviews in Fluorescence (Geddes, C. D., Ed.) pp 17-43, Springer International Publishing. (19) Geißler, D., Charbonnière, L. J., Ziessel, R. F., Butlin, N. G., Löhmannsröben, H. G., and Hildebrandt, N. (2010) Quantum Dot Biosensors for Ultrasensitive Multiplexed Diagnostics. Angew. Chem. Int. Ed. 49, 1396-1401. (20) Cardoso Dos Santos, M. and Hildebrandt, N. (2016) Recent developments in lanthanideto-quantum dot FRET using time-gated fluorescence detection and photon upconversion. TrAC Trends Anal. Chem. 84, 60-71. (21) Charbonnière, L. J. and Hildebrandt, N. (2008) Lanthanide Complexes and Quantum Dots: A Bright Wedding for Resonance Energy Transfer. Eur. J. Inorg. Chem., 3231-3231. (22) Geißler, D., Linden, S., Liermann, K., Wegner, K. D., Charbonnière, L. J., and Hildebrandt, N. (2014) Lanthanides and Quantum Dots as Förster Resonance Energy Transfer Agents for Diagnostics and Cellular Imaging. Inorg. Chem. 53, 1824-1838. (23) Hildebrandt, N. (2011) Biofunctional Quantum Dots: Controlled Conjugation for Multiplexed Biosensors. ACS Nano 5, 5286-5290. (24) Wegner, K. D., Jin, Z., Lindén, S., Jennings, T. L., and Hildebrandt, N. (2013) QuantumDot-Based Förster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples. ACS Nano 7, 7411-7419. (25) Wegner, K. D., Lindén, S., Jin, Z., Jennings, T. L., Khoulati, R. e., van Bergen en Henegouwen, P. M. P., and Hildebrandt, N. (2014) Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection. Small 10, 734-740. (26) Qiu, X., Wegner, K. D., Wu, Y.-T., van Bergen en Henegouwen, P. M. P., Jennings, T. L., and Hildebrandt, N. (2016) Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET. Chem. Mater. 28, 8256-8267. (27) Bhuckory, S., Mattera, L., Wegner, K. D., Qiu, X., Wu, Y. T., Charbonniere, L. J., Reiss, P., and Hildebrandt, N. (2016) Direct conjugation of antibodies to the ZnS shell of quantum dots for FRET immunoassays with low picomolar detection limits. Chem. Commun. 52, 14423-14425. (28) Mattera, L., Bhuckory, S., Wegner, K. D., Qiu, X., Agnese, F., Lincheneau, C., Senden, T., Djurado, D., Charbonniere, L. J., Hildebrandt, N., and Reiss, P. (2016) Compact quantum dotantibody conjugates for FRET immunoassays with subnanomolar detection limits. Nanoscale 8, 11275-11283. 22 ACS Paragon Plus Environment

Page 22 of 24

Page 23 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Baniukevic, J., J.Kirlyte, Ramanavicius, A., and Ramanaviciene, A. (2012) Comparison of Oriented and Random Antibody Immobilization Techniques on the Efficiency of Immunosensor. Procedia Eng. 47, 837-840. (30) Baniukevic, J., Kirlyte, J., Ramanavicius, A., and Ramanaviciene, A. (2013) Application of oriented and random antibody immobilization methods in immunosensor design. Sens. Actuator B-Chem. 189, 217-223. (31) Bilan, R., Brazhnik, K., Chames, P., Baty, D., Nabiev, I., and Sukhanova, A. (2015) Oriented Conjugates of Single-domain Antibodies and Fluorescent Quantum Dots for Highly Sensitive Detection of Tumor-associated Biomarkers in Cells and Tissues. Phys. Procedia 73, 228-234. (32) Brazhnik, K., Nabiev, I., and Sukhanova, A. (2014) Advanced Procedure for Oriented Conjugation of Full-Size Antibodies with Quantum Dots. Quantum Dots: Applications in Biology (Fontes, A., Santos, B. S., Eds.) pp 55-66, Springer New York. (33) Brazhnik, K., Nabiev, I., and Sukhanova, A. (2014) Oriented Conjugation of SingleDomain Antibodies and Quantum Dots. Quantum Dots: Applications in Biology (Fontes, A., Santos, B. S., Eds.) pp 129-140, Springer New York. (34) Makaraviciute, A. and Ramanaviciene, A. (2013) Site-directed antibody immobilization techniques for immunosensors. Biosens. Bioelectron. 50, 460-471. (35) Sukhanova, A., Even-Desrumeaux, K., Kisserli, A., Tabary, T., Reveil, B., Millot, J.-M., Chames, P., Baty, D., Artemyev, M., Oleinikov, V. et al. (2012) Oriented conjugates of singledomain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine 8, 516-525. (36) Tajima, N., Takai, M., and Ishihara, K. (2011) Significance of Antibody Orientation Unraveled: Well-Oriented Antibodies Recorded High Binding Affinity. Anal. Chem. 83, 19691976. (37) Tasso, M., Singh, M. K., Giovanelli, E., Fragola, A., Loriette, V., Regairaz, M., Dautry, F., Treussart, F., Lenkei, Z., Lequeux, N. et al. (2015) Oriented Bioconjugation of Unmodified Antibodies to Quantum Dots Capped with Copolymeric Ligands as Versatile Cellular Imaging Tools. ACS Appl. Mater. Interfaces 7, 26904-26913. (38) Zhang, B., Yu, J., Liu, C., Wang, J., Han, H., Zhang, P., and Shi, D. (2016) Improving detection sensitivity by oriented bioconjugation of antibodies to quantum dots with a flexible spacer arm for immunoassay. RSC Adv. 6, 50119-50127. (39) Pons, T., Medintz, I. L., Wang, X., English, D. S., and Mattoussi, H. (2006) SolutionPhase Single Quantum Dot Fluorescence Resonance Energy Transfer. J. Am. Chem. Soc. 128, 15324-15331. (40) Jennings, T. L., Becker-Catania, S. G., Triulzi, R. C., Tao, G., Scott, B., Sapsford, K. E., Spindel, S., Oh, E., Jain, V., Delehanty et al. (2011) Reactive Semiconductor Nanocrystals for Chemoselective Biolabeling and Multiplexed Analysis. ACS Nano 5, 5579-5593. (41) Lee, S. F. and Osborne, M. A. (2009) Brightening, Blinking, Bluing and Bleaching in the Life of a Quantum Dot: Friend or Foe? ChemPhysChem 10, 2174-2191. (42) Noble, J. E. and Bailey, M. J. (2009) Quantitation of protein. Methods Enzymol. 463, 7395. (43) Wegner, K. D., Lanh, P. T., Jennings, T., Oh, E., Jain, V., Fairclough, S. M., Smith, J. M., Giovanelli, E., Lequeux, N., Pons, T., and Hildebrandt, N. (2013) Influence of Luminescence Quantum Yield, Surface Coating, and Functionalization of Quantum Dots on the Sensitivity of Time-Resolved FRET Bioassays. ACS Appl. Mater. Interfaces 5, 2881-2892. 23 ACS Paragon Plus Environment


(44) Bhuckory, S., Lefebvre, O., Qiu, X., Wegner, K., and Hildebrandt, N. (2016) Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen. Sensors 16, 197.


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