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Polymer entrapped quantum dots ‘papaya particles’ improve immunoassay sensitivity in serum Andrea Ranzoni, Anniek den Hamer, Tomislav Karoli, Joseph Buechler, and Matthew A. Cooper Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b00762 • Publication Date (Web): 13 May 2015 Downloaded from http://pubs.acs.org on May 16, 2015

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Analytical Chemistry

Polymer entrapped quantum dots ‘papaya particles’ improve immunoassay sensitivity in serum Andrea Ranzoni†, Anniek den Hamer†, Tomislav Karoli†, Joseph Buechler‡, Matthew. A. Cooper†,* †) Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, 4072, Australia ‡) ALERE Inc. 9975 Summers Ridge Rd, San Diego, CA 92121, USA KEYWORDS (Dengue fever, Quantum Dots, Human serum, Serotyping, Polymer chemistry, Immunoassay, Diagnostics, High sensitivity.

ABSTRACT: Fluorescent labels are widely employed in biomarker quantification and diagnostics, however they possess narrow Stokesshifts and can photo-bleach, limiting multiplexed detection applications and compromising sensitivity. In contrast, quantum dots do not photobleach and have much wider Stokes-shifts, but a paucity of robust surface attachment chemistries for bio-conjugation has limited their uptake in biomedical diagnostics. We report a novel class of biofunctional fluorescent labels based on trapping of ~104 quantum dots within a core nanoparticle. The doped particles act as scaffolds for generation of a multi-layered shell comprised of a functionalized hydrophilic polymer with covalently attached receptors for analyte capture. These constructs, which conceptually resemble a papaya fruit, are chemically stable, remain mono-dispersed for > 6 months in buffer, and show utility in immunoassay applications. Using monoclonal antibody fragments against non-structural protein dengue NS1, an early biomarker for dengue fever, antibody immobilization capacity was 75-fold higher compared to traditional carbodiimide protein coupling. In the model dengue immunoassay, we observed a 15-fold lower limit of detection and 4-fold higher fluorescence intensity with the ‘papaya particles’ compared to current ‘best-in-class’ commercial reagents. Direct deployment in human serum allowed sensitive detection of different NS1 serotypes with lower limits of detection within the clinically relevant range (1-10 ng/mL) and sufficient specificity for identification of the dengue serotype was achieved for concentrations >10 ng/mL (DV1-3) and >50 ng/mL (DV4). The combination of chemical and physical stability, high binding capacity combined with the intrinsic advantages of quantum dots may enable more simple, robust diagnostic assays in the future.

Technologies to detect biomarkers directly in complex matrices are widely employed in life sciences and medical diagnostics. A common strategy to facilitate detection involves labelling of the biomarkers with fluorescent or bioluminescent reporter ligands1. Assay formats based on organic dyes delivered multiplexed detection of protein biomarkers and molecular interactions between proteins and peptides2-4, however the unique spectral fingerprint of fluorescent dyes necessitates dedicated optics for each fluorophore to enable multiplexed readouts and maximize detection sensitivity 2,5. In contrast, semiconductor quantum dots (QDs) exhibit superior photophysical properties compared to organic and small molecule dyes: one single light source can trigger high efficiency emission at a wavelength correlated to the size of the quantum crystals6. Biotechnologies based on QDs would therefore require more compact readouts and would yet deliver brightness superior to standard fluorophores. Extensive multiplexing has been demonstrated, resulting in QDs being widely praised as ideal candidates for next generation labels. However, QDs have yet to find widespread adoption in commercial diagnostic assays. The lack of simple, robust methods for stable bio-functionalization has confined their use to the research sector, principally in academia7,8. QDs are synthesized from organometallic precursors and are inherently hydrophobic. Ligand exchanging the original hydrophobic surface capping with bifunctional ligands imparts solubility with concomitant availability of a chemical moiety for bioconjugation 9. However, these approaches are limited, mainly involve non-specific physisorption (e.g. oleic acid, phosphatidylethanolamine, amphiphilic saccarides, histidine–rich epitopes or polyhistdidine), as reviewed in 10. Dative thiol bonds with mercaptoalkylacids or dihydrolipoic acid are not as ordered or stable as

those found in self-assembled monolayers on gold particles and planar gold surfaces. Accordingly, these approaches do not allow precise preparation of QD bioconjugates with fine control over the ratio of biomolecules per QD and subsequent orientation of the analyte capture reagents. Hence, much work still needs to be done to achieve reproducible and robust surface functionalization and bioconjugation with QDs and detection of protein biomarkers directly in complex matrices is still in its formative stage 11-14 . An alternative approach relies on doping nanoparticles with a fluorescent agent. An individual nanoparticle can trap several thousand dye molecules, therefore effectively amplifying each labelling event15-19. Park et al. have built zwitterionic, physisorbed streptavidin-QDs around a biotinylayed agarose core nanoparticle; the zwitterionic surface coating causing self-assembly of QDs to amplify signal in a multi-step assay 20. Others have enhanced detection sensitivity by combining enzymatic amplification with fluorescent detection 21. Introducing a polymeric support to physically trap the QDs enables more controlled bioconjugation, thus circumventing the complications related to direct biofunctionalization of QDs. However the most routinely used methods use carbodiimide chemistry or the high affinity interaction between biotin and streptavidin, whose limitations have been recently highlighted22,23. Examples of QD nanoparticle-based immunoassay working directly in complex matrices are rare20,21,24,25. In this work, we introduce a novel class of particles decorated with a three-dimensional hydrogel capable of robust and stable trapping of semiconductor QDs and affinity probes on the nanoparticle surface (Figure 1). The resulting construct can be conceptually compared to a papaya fruit; QDs embedded within polystyrene nanoparticles constitute the seeds within the core of the fruit, and the bioactive QD-doped hydrogel represents the pulp

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of the papaya. To evaluate the performance of the particles in a clinically relevant immunoassay, we developed an assay for dengue infection. Dengue is an infectious disease borne by Aedes mosquitos with almost a third of the world population at risk of infection 26, progressively expanding towards non-tropical regions, and now endemic in more than 125 countries. With the global distribution constantly spreading, the associated clinical burden is on the rise26,27. Currently, no cure or vaccine is available to treat the disease, which can manifest itself in four serotypes (DENV1-4) 28. Infection by one serotype induces life-long immunity against reinfection by the same serotype, but only partial protection from another serotype. Subsequent infection by different strains leads to dramatic changes of the pathophysiology of the disease, resulting in potentially fatal haemorrhagic fever (DHF) or dengue shock syndrome (DSS) 29. The gold standard for serotyping of dengue involves amplification of the viral RNA30,31, however a biomarker-based immunoassay with sufficient sensitivity and viral subtype sero-specificity in a format amenable to resource-limited settings would contribute to rapid diag-

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nosis in the field. One of the most well-established early biomarkers is non-structural protein NS1, released in the bloodstream during viral replication.Hydrogel-mediated loading of QDs resulted in brightness superior to ‘best-in-class’ fluorescent nanoparticles and in 75-fold higher conjugation of capture probes compared to conventional carbodiimide chemistry. In a direct comparison in a sandwich immunoassay, the fluorescent papaya particles outperformed commercial analogues, delivering 15-fold lower limit of detection. The particles exhibited longterm stability in aqueous solutions, with no appreciable loss of bound QDs after six months at room temperature, compared to previous approaches that resulted in rapid dissociation of QDs after few days32-34. Figure 1a summarizes the synthesis: polystyrene nanoparticles (200 nm or 500 nm in diameter) underwent swelling in organic solvent to facilitate incorporation of QDs in the bulk volume. The nanoparticles and the QDs were exposed to ultrasound to ensure complete dispersion in a 5% solution of toluene in butanol. Diffusion-driven migration of the QDs within the swollen polymer resulted in the QDs progressively ac

Figure 1. Schematic of the multi-layer surface molecular architecture and covalent linkages between the ‘papaya particle’ surface, quantum dots and monoclonal antibody fragments. a) Quantum dots doped within the bulk volume of the polystyrene nanoparticle are subsequently delocalized in a multi-layered approach within a PDEC-dextran network. b) An assay format for sensitive serotype-specific detection of NS1 protein. Cross-reactive antibody fragments are immobilized on the sensor surface. The target NS1 is captured by the immobilized antibodies and the ‘papaya particles’ complete the sandwich generating high-intensity fluorescence for target quantification.


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cumulating within the nanoparticles. Addition of -mercaptopropyltrimetoxysilane (MPTS) and traces of aqueous solvent triggered polymerization and therefore helped to physically constrain the QDs. The papaya-like nanoparticles were then washed thoroughly in a 1:1 mixture of ethanol and toluene and the supernatant was used to estimate the QD incorporation efficiency. A reference curve was obtained by serially diluting the stock solution of QDs, with the measured fluorescence intensity from the supernatant enabling quantification of the QDs not incorporated within the nanoparticles (Supplementary Information). For low (100 to 1000-fold) molar excesses of QD/nanoparticle, the incorporation efficiency was close to 100%, whereas for > 10 4 molar excesses the capture efficiency decreased, indicating attainment of maximum doping. When using a molar excess of 2.5 x 104 QD/nanoparticle we estimated an efficiency of incorporation of 92 ± 9% corresponding to (2.2 ± 0.2) x 10 4 QDs for each 500 nm nanoparticle. We observed a 40% reduction in fluorescent intensity for QDs trapped as “papaya seeds” vs. equivalent absolute numbers of QDs in dispersed free solution, which was most likely due to reduced emission efficiency due to electron transfer from neighboring atoms in the scaffolds. The emission spectra showed no broadening nor wavelength shift, indicating that the QDs did not significantly aggregate, in agreement with previous findings 18. The density of sulfhydryl groups available post-polymerization was quantified by means of Ellman’s assay (Supplementary Information). We estimated that (2.58 ± 0.18) x 106 sulfhydryl groups were available for binding on each nanoparticle, with minimal batch-to-batch variability (Supplementary Information). If one approximates the core polystyrene nanoparticles as spheres with negligible surface roughness, such value corresponds to 3.28 ± 0.24 sulfhydryl groups per nm2, close to maximum molecular packing on the surface. Subsequently, hydrophobic quantum dots were derivatized with sulfhydryl-reactive dextran derivative (2-(pyridinyldithio)ethylcarbamoyl- or PDECdextran 35) introduced in a one-step reaction together with the thiolated nano-scaffolds. The low pKa of the conjugate acid for the leaving group prevents cross-linking between dextran molecules 35 unless mediated by a polyvalent binding agent (i.e. in this case the quantum dot). The ‘one-pot’ incubation leads to mutual cross-linking: the PDEC dextran generates a hydrophilic layer on the nanoparticle surface and cross-links to the metal surface of the QDs by generating a dative bond that displaces the hydrophobic coating (Figure 1a). As a result, the polymer-entrapped QDs were colloidally stable in aqueous solutions. The reaction was limited by the number of the QDs, therefore all the conditions we tested resulted in immobilization efficiencies higher than 99%. We measured 980 ± 190 QD on the external surface of each nanoparticle, in good agreement with the theoretical average (~800), under the assumptions of unitary efficiency and no quenching. These assumptions are justified in the Supporting Information. Before addressing strategies for biofunctionalization, we first examined particle stability and brightness, benchmarked against standard carbodiimide chemistry for the formation of an amide bond between the nanoparticle surface and the QDs (amide bond in Fig. 2a) 33 and direct coupling of hydrophobic QDs on a thiol-modified surface (sulfhydryl/metal in Fig. 2a) 32,34. These literature couplings and particle characterization are detailed in the Supplementary Information. We then periodically measured the fluorescence intensity of each batch, storing each sample in

Figure 2. a) Long-term stability of the ‘papaya particles’ in aqueous buffer. Quantum dots bound by means of an amide bond or directly interacting with a sulfhydryl proved unstable in aqueous solutions after few days. b) Absolute fluorescence intensity of the papaya-like nanoparticles compared to commercially available dye-doped fluorescent nanoparticles. The number of nanoparticles was calculated according to manufacturer’s specifications. c) 75-fold higher density of affinity probes immobilized onto the surface of the papaya-like nanoparticles by means of PDEC dextran.



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aqueous buffer between different measurements and performing a washing step to remove QDs that had potentially became unbound from the core particle. We have observed a progressive leaching of QDs for both traditional literature approaches, with loss of up to 80% of the bound dots in less than one week. In vivid contrast, our ‘papaya particles’ remained stable after several months storage in aqueous buffers with no measurable leakage of QDs. We then compared 500 nm diameter ‘papaya particles’ to fluorescent particles purchased from Bangs Laboratories and from Kisker Biotech. 200 nm diameter ‘papaya particles’ were also compared with commercial particles of identical diameter: Life Technologies™ FluoSpheres®, containing approximately 105 equivalents of fluorescent dye per nanoparticles according to manufacturer’s specifications. To our knowledge, this family of fluorescent nanoparticles is the brightest currently commercially available. Measured fluorescence intensity under optimal settings using a microplate reader (Perkin Elmer EnVision®) gave 4050% higher signal for ‘papaya particles’ compared to equal numbers of commercial nanoparticles (Figure 2b). The encapsulation of the nanoparticles with large excess of hydrophilic PDEC dextran utilized only a fraction of the available 2-pyridinyldithio reactive groups for surface immobilization, therefore enabling facile immobilization of affinity capture probes. For the dengue immunoassay, the affinity probes were

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derived from two recombinant antibodies that cross-react with all four dengue NS1 serotypes. The antibodies consisted of the entire kappa chain, and the variable and CH1 domain of the heavy chain. The last amino acid of the kappa chain were mutated to a serine, which lead to a free cysteine at the end of the heavy chain constant region. The sulfhydryl at the hinge region of the monoclonal antibody fragments (mFAbs) could react with the PDEC moiety, thus enabling precise cross-linking without affecting the high-affinity epitope. Hence, the biofunctionalization step did not require additional coupling reagents and simple mixing completed the assembly of the surface molecular architecture of immunoassay-ready ‘papaya particles’. Successful conjugation was verified by means of a bicinchoninic acid (or BCA) assay to quantify the level of protein loading (Figure 2c). Conventional methods for biofunctionalization of nanoparticles are based on carbodiimide chemistry (“Carbodiimide” in Figure 2c), which directly couples the affinity probes to the nanoparticle surface. Consequently, the maximum density of capture probes that can be immobilized corresponds to an approximate monolayer of protein. In our case, the PDEC-dextran network self-assembled onto the core nanoparticles serves to enhance the available surface area for immobilization in a hydrophilic polymer background. When no QDs are immobilized externally, the polymer network is “thin” and corresponds to a monolayer of PDEC dextran on the nanoparticle surface (“Mono-layer” in Figure 2c).

Figure 3. Dose-response curves in buffer, demonstrating serotype-specific detection of NS1 protein in buffer.


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When QDs are incorporated in the outer layer, they act as anchoring point for further dextran monomers, thus “seeding” multi-layered self-assembly of the dextran with QDs strongly trapped within. The number of QDs in the reaction hence limits and controls the polymer thickness (“Multi-layer” in Figure 2c). We prepared three independent batches for each approach, using the same input concentration of antibody fragments and corresponding to several theoretical protein monolayers. We observed a progressive increase in the number of affinity probes, with 75-fold increase in the number of capture probes (0.21 ± 0.01 mFAbs/nm2) compared to the carbodiimide approach (0.0029 ± 0.0004 mFAbs/nm2). In consideration of rapid immunoassay diagnostic performance, we note that the time constant regulating the depletion of a target biomarker from a solution is inversely proportional to the antibody loading on the nanoparticles in a Langmuir model 36. In addition, polymeric coatings are very effective in mitigating non-specific interactions arising from abundant endogenous interfering agents present in biological fluids that also serve to slow the transport of the target biomarker to the capture moieties 27,36. Consequently, the ‘papaya particles’ are particularly suited for demanding affinity-based biosensing applications, where minute biomarker concentrations need to be rapidly detected directly from biological fluids. To probe the clinical relevance of our approach, we developed a biomarker-based assay to serotype dengue fever in human serum. The assay protocol is sketched in Figure 1b. The mFAb immobilized on the surface was capable of recognizing all four dengue NS1 serotypes with high affinity. The serum sample was incubated with a functionalized surface to sequester the dengue NS1 antigen. The

functional nano-scaffolds bound the immobilized biomarker in a sandwich configuration, and after a washing step, the detected fluorescence intensity was correlated with the concentration of biomarker in solution. We first validated our assay and demonstrated dose-response curves in buffer (Figure 3) for each dengue serotype. We could demonstrate a lower limit of detection (LoD) of 0.5 ng/mL (DENV-1), 0.2 ng/mL (DENV-2), 0.8 ng/mL (DENV-3) and 1 ng/mL (DENV-4), well below reported levels of circulating NS1 in patients affected from primary or secondary infection (reference values of circulating NS1: 13 ng/mL – 1880 ng/mL for patients infected with DENV-1 and 4 ng/mL – 266 ng/mL for patients infected with DENV-2 in a cohort study of 225 patients) 37. We were able to demonstrate sensitive and selective detection of each dengue serotype with minimal cross talk between serotypes. The most common clinical serotype (DENV2) was detected with the highest sensitivity and the developed assay has a dynamic range of two to three orders of magnitude for each serotype. Direct deployment in human serum lead to dose-response curves comparable to buffer for all dengue serotypes (Figure 4). The assay retained serotyping specificity and lower LoD below 1 ng/mL for each serotype, once again well within the clinically relevant range38. Partial serotyping has been previously reported for dengue, however the reported sensitivities varied more than two orders of magnitude between different serotypes and the input concentration of biomarker was not fully quantified39. To our knowledge, this is the first example of a biomarker-based immunoassay with sufficient serotype specificity to be able to discriminate dengue serotype directly in complex fluids at clinically relevant levels.

Figure 4. Dose-response curves in neat human serum, demonstrating serotype-specific detection of NS1 protein.



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The assay format was then used to further investigate the impact of the polymer network and superior brightness on analytical performances. In an attempt to benchmark the ‘papaya particles’ with “best-in-class” reagents, we coated Life Technologies™ FluoSpheres® with anti-fouling surface chemistry optimized elsewhere23 for detection of NS1 protein in human serum. Papayalike nanoparticles 200 nm in diameter were prepared with an antibody density of 0.23 ± 0.01 mFAbs/nm2. Assuming a maximally packed perfect monolayer of mFAbs on the nanoparticle surface, this value corresponds to approximately 8 monolayers (approximate size of a mFAb 8x4 nm2). The FluoSpheres, approximately 40% less bright, had an antibody density of ~10-fold less; 0.021 ± 0.025 mFAbs/nm2.The shift in dose-response curves clearly highlights the improved assay performances, resulting in 13-fold lower limit of detection. Fig. 5b shows the absolute fluorescence intensity for selected concentrations and demonstrates increased number of binding events mediated by the high density of affinity probes, which yielded 6-fold higher fluorescence signal in biological immunoassays. No dose-response curve could be demonstrated for carbodiimide chemistry, as the analyte-dependent signal was completely masked by the high non-specific background (See Supplementary Information). Tight packing of bioactive molecules on a nanoparticle surface has been shown to impair affinity of a capture reagent for the cognate target, since overcrowding induces steric hindrance preventing access to the affinity epitope 22. Less than 4% of the antibodies immobilized using by carbodiimide chemistry remained active after direct coupling to a nanoparticle surface 22. We developed a “depletion assay” to quantify the number of active antibodies per nanoparticle (Supplementary Information). A controlled number of nanoparticles (0.4 fmol) was incubated with known amounts of NS1 (50 ng and 5 ng) until the capture reaction reached equilibrium. After a washing step, the supernatant was used as input for an ELISA and the resultant signal compared with a calibration curve. Samples with only 5 ng of NS1 protein were completely depleted with no measurable signal in the ELISA assay. Samples containing 50 ng of NS1 protein led to >65% of antibodies remaining active. These findings indicate that delocalization of capture probes within a hydrophilic threedimensional structure helps preserving the high affinity towards the target. In conclusion, we describe a novel method for immobilization of semiconductor quantum dots within a hydrophilic bioactive polymer network. Each step of the self-assembly of the molecular architecture was characterized, thus delivering a method with quality control steps for minimal batch-to-batch variability. We demonstrated exceptional storage stability of particles with much brighter fluorescence than commercial fluorescent nanoparticles, and 75-fold higher loading capacity for affinity probes. The delocalization of the affinity probes within a hydrophilic polymer possibly also prevents overcrowding at the surface, resulting in a 15fold higher sensitivity when benchmarked against ‘best-in-class’ commercial nanoparticles. Direct deployment in complex biological fluids resulted in a sensitive immunoassay capable of detecting NS1 levels within the clinically relevant range. Given the high cross reactivity between different dengue strains and other flaviviruses and the anamnestic antibody response in secondary infections, serotype discrimination with existing serological assays is unfeasible. Nucleic acid amplification of the viral genome is

Figure 5. a) Dose-response curve for DENV4-NS1 in buffer obtained with 200 nm ‘papaya-like nanoparticles’ and Life Technology F8811 functionalized with anti-fouling surface chemistry (See Supplementary Information). The ‘papaya particle’ gave a 15-fold lower limit of detection (IC50papaya=25 ng/mL, IC50LifeTech=340 ng/mL) and b) a 6fold superior intensity.

currently the only method with enough viral subtype sero-specificity, however this approach is hardly amenable to resource-limited settings. The exquisite specificity of the mFAbs within our nano-constructs allows for rapid serotyping of dengue fever from a serum sample and will enable rapid, cost-effective immunoassays in the field. Direct testing of NS1-carrying dengue infected mosquitoes in the field would also aid close monitoring of the impact of measures of vector control. The robustness, scalability and superior assay performance render this nanotechnology suited for utilization in a wide range of applications. Hybrid magnetic nanoparticles with QDs on the surface can be easily prepared for multi-modal imaging and theranostics 40-43. Efficient coating of sensor surfaces would minimize biofouling and extend the sensor lifetime for ex vivo applications in whole blood while simultaneously accelerating binding kinetics with target molecules44-46. Enhanced kinetics will additionally enable rapid depletion of patient samples, strongly advocated for high sensitivity biosensing at the point-of-care47-50.


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Preparation of 4‐nitrophenyl carbonated dextran. To a solution of dextran T70 (180 mg) in 1.8 mL of anhydrous pyridine and 2 mL of anhydrous DMSO were added nitrophenylchloroformate (90mg) and catalytic amount of 4‐dimethylaminopyridine. The solution was gently stirred for 5 hours at 0˚C before adding a mixture of 1:1 methanol:diethyl ether. After overnight precipitation the solid content was washed three times with 1:1 methanol:diethylether solution using a filter under vacuum. The precipitate was dried under high vacuum yielding 110mg of product. 1H NMR (600 MHz, d6-DMSO) selected δ: 8.32 (br s, 2H of 4-nitrophenyl), 7.56 (br s, 2H of 4-nitrophenyl). Degree of incorporation was estimated as 9 mol% on a per glucose unit basis by using line-shape analysis to correct for the water interference at 3.38 ppm. The degree of incorporation corresponds well to that reported in the literature [Ref. 30]. Preparation 2‐(pyridinyldithio)ethyl carbomoyl dextran. 4‐nitrophenyl carbonated dextran (110mg) and 2‐(pyridinyldithio)ethaneamine (PDEA, 45 mg) were dissolved in 0.42 mL of anhydrous pyridine. 1.4 mL DMSO and 28 μL of methylmorpholine were added and the mixture was stirred O/N at RT. The mixture was precipitated by slowly adding methanol/diethyl ether (1:1) mixture while stirring. The precipitates were washed three times with 1:1 methanol:diethylether solution using a filter under vacuum. The compound was dried under high vacuum for ~ 3 hours, yielding 76 mg of 2‐(pyrindinyldithio) ethyl carbamoyl dextran (PDEC dextran). 1 H NMR (600 MHz, d6-DMSO) selected δ: 8.46 (br s, 1H pyridinyl), 7.84 (br s, 1H pyridinyl), 7.78 (d, 1H pyridinyl), 7.25 (br s, 1H pyridinyl). Degree of incorporation was estimated as 1 mol% on a per glucose unit basis by using line-shape analysis to correct for the water interference at 3.32 ppm. The degree of incorporation corresponds well to that reported in the literature [Ref. 30]. Bio-functionalized ’papaya’ nanoparticles. Step1: incorporation QDs by swelling. The method was adapted from Ref. 18. 100µL of Polybeads-amino (200 nm or 500 nm) were washed twice with butanol by centrifugation (2 minutes at 13k rcf). The nanoparticles were suspended in a mixture of toluene-butanol 5:95 and placed in a sonic bath for 60 s. A solution of 500 pmol of PL-QD-O-590 in toluenebutanol 5:95 was added to the nanoparticles and incubated for 30 minutes under gentle shaking in darkness. -mercaptopropyltrimetoxysilane (MPTS, 10 µmol) was added to the solution and incubated for 20 minutes before adding traces of deionized water. The nanoparticles were washed three times in absolute ethanol. Step2: Generation of 3D dextran network. PDEC-Dextran 70 KDa (0.1 mg) and Q21711MP (40 pmol) are added to the sample obtained at step 1. After 4 hours of incubation, the nanoparticles were washed in absolute ethanol and resuspended in PBSP buffer (10mM PBS pH 7.4, 0.1% Pluronic F127). Step3: Bio-functionalization. The mutated serine at the end of the kappa chain of the mFAbs introduced a free cysteine capable of reacting with the 2-pyridinyldithio moiety without need of coupling agents. Antibody fragments against dengue nonstructural protein (NS1) were added to the nanoparticles (12.5 µg every mg of nanoparticles) and incubated overnight at 4˚C. The nanoparticles are washed three times and stored in PBSP buffer at a concentration of 10 mg/mL.

Surface functionalization. NUNC Maxisorp 96-well plates (442404-21) were incubated overnight with 100 µL/well of mFAb 351 (5 µg/mL in carbonate buffer 50 mM pH 8.5), cross-reactive against all four dengue serotypes. The wells were washed with PBSP buffer and subsequently blocked with KPL buffer (KPL5082-01). The plates were stored at 4˚C until use. Assay protocol. Functionalized NUNC Maxisorp 96-well were incubated with NS1 protein serially diluted in human serum (or PBSP buffer). After a brief washing step, the nano-particles were

added to the well and incubated for 1 hour to allow for ligation to the surface. The unbound particles were removed via a washing step with PBSP buffer and the fluorescent intensity was read with Perkin Elmer EnVision plate reader. Depletion assay. DV2 NS1 protein was diluted in PBSP buffer (10mM PBS, pH 7.4, 0.1% Pluronic F127). 0.5 fmol of nanoparticles were incubated with 0.1 mL of NS1 protein. After 2h at 37 C, the nano-particles were centrifugated and the supernatant was used as input for an ELISA assay. Monoclonal Fab 351 was immobilized overnight on a NUNC Maxisorp 96-well plate (442404-21) and a serial dilution of NS1 protein was incubated for 1.5 h at 37C. After three washes with PBSP, a HRP-mFAb against DV2 NS1 conjugate developed in house was used to determine the levels of bound NS1.

A detailed description of the synthesis of PDEC dextran including NMR spectra, quantification of the number of QDs incorporated by swelling, quantification of the number of sulfhydryl groups on the surface of the nanoparticles, quantification of the number of QDs on the outer layer of the papayas, evaluation of the density of antibody fragments on the surface of the papaya-like nanoparticles, stability of the papaya-like nanoparticles in aqueous buffers, preparation of the literature surface chemistries used to benchmark stability, pre-assay preparation of the sensor surface, assay protocol, depletion assay protocol. “This material is available free of charge via the Internet at http://pubs.acs.org.”

* Address correspondence to Prof. Matthew A. Cooper [email protected]

The authors would like to acknowledge The University of Queensland for partially funding this work.

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