Self-assembled quantum dot-peptide bioconjugates for selective intracellular delivery

We demonstrate the use of self-assembled luminescent semiconductor quantum dot (QD)-peptide bioconjugates for the selective intracellular labeling of several eukaryotic cell lines. A bifunctional oligoarginine cell penetrating peptide (based on the HIV-1 Tat protein motif) bearing a terminal polyhistidine tract was synthesized and used to facilitate the transmembrane delivery of the QD bioconjugates. The polyhistidine sequence allows the peptide to self-assemble onto the QD surface via metal-affinity interactions while the oligoarginine sequence allows specific QD delivery across the cellular membrane and intracellular labeling as compared to nonconjugated QDs. This peptide-driven delivery is concentration-dependent and thus can be titrated. Upon internalization, QDs display a punctate-like staining pattern in which some, but not all, of the QD signal is colocalized within endosomes. The effects of constant versus limited exposure to QD-peptide conjugates on cellular viability are evaluated by a metabolic specific assay, and clear differences in cytotoxicity are observed. The efficacy of using peptides for selective intracellular delivery is highlighted by performing a multicolor QD labeling, where we found that the presence or absence of peptide on the QD surface controls cellular uptake.     

Photoabsorption and resonance energy transfer phenomenon in CdTe-protein bioconjugates: an insight into QD-biomolecular interactions

Luminescent quantum dots (QDs) possess unique photophysical properties, which are advantageous in the development of new generation robust fluorescent probes based on Forster resonance energy transfer (FRET) phenomena. Bioconjugation of these QDs with biomolecules create hybrid materials having unique photophysical properties along with biological activity. The present study is aimed at characterizing QD bioconjugates in terms of optical behavior. Colloidal CdTe QDs capped with 3-mercaptopropionic acid (MPA) were conjugated to different proteins by the carbodiimide protocol using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS). The photoabsorption of these QD-protein bioconjugates demonstrated an effective coupling of electronic orbitals of constituents. A linear variation in absorbance of bioconjugates at 330 nm proportionate to conjugation suggests a covalent attachment as confirmed by gel electrophoresis. A red shift in the fluorescence of bovine serum albumin (BSA) due to conjugation inferred a decrease in Stokes shift and solvent polarization effects on protein. A proportionate quenching in BSA fluorescence followed by an enhancement of QD fluorescence point toward nonradiative dipolar interactions. Further, reduction in photobleaching of BSA suggests QD-biomolecular interactions. Bioconjugation has significantly influenced the photoabsorption spectrum of QD bioconjugates suggesting the formation of a possible protein shell on the surface of QD. The experimental result suggests that these bioconjugates can be considered nanoparticle (NP) superstructures for the development of a new generation of robust nanoprobes.     

Aromatic aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live cell imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.     

Intracellular delivery of quantum dot-protein cargos mediated by cell penetrating peptides

We utilize cell penetrating peptide functionalized QDs as specific vectors for the intracellular delivery of model fluorescent protein cargos. Multiple copies of two structurally diverse fluorescent proteins, the 27 kDa monomeric yellow fluorescent protein and the 240 kDa multichromophore b-phycoerythrin complex, were attached to QDs using either metal-affinity driven self-assembly or biotin-Streptavidin binding, respectively. Cellular uptake of these complexes was found to depend on the additional presence of cell-penetrating peptides within the QD-protein conjugates. Once inside the cells, the QD conjugates were mostly distributed within endolysosomal compartments, indicating that intracellular delivery of both QD assemblies was primarily driven by endocytotic uptake. Cellular microinjection of QD-fluorescent protein assemblies was also utilized as an alternate delivery strategy that could bypass the endocytic pathway. Simultaneous signals from both the QDs and the fluorescent proteins allowed verification of their colocalization and conjugate integrity upon delivery inside live cells. Due to their intrinsic fluorescence properties, this class of proteins provides a unique tool to test the ability of QDs functionalized with cell penetrating peptides to mediate the intracellular delivery of both small and large size protein cargos. Use of QD-peptide/fluorescent protein vectors may make powerful tools for understanding the mechanisms of nanoparticle-mediated drug delivery.     

Aptamer-functionalized quantum dots as theranostic nanotools against cancer and bacterial infections: A comprehensive overview of recent trends

Aptamers (Apts) are synthetic nucleic acid ligands that can be engineered to target various molecules, including amino acids, proteins, and pharmaceuticals. Through a series of adsorption, recovery, and amplification steps, Apts are extracted from combinatorial libraries of synthesized nucleic acids. Using aptasensors in bioanalysis and biomedicine can be improved by combining them with nanomaterials. Moreover, Apt-associated nanomaterials, including liposomes, polymeric, dendrimers, carbon nanomaterials, silica, nanorods, magnetic NPs, and quantum dots (QDs), have been widely used as promising nanotools in biomedicine. Following surface modifications and conjugation with appropriate functional groups, these nanomaterials can be successfully used in aptasensing. Advanced biological assays can use Apts immobilized on QD surfaces through physical interaction and chemical bonding. Accordingly, modern QD aptasensing platforms rely on interactions between QDs, Apts, and targets to detect them. QD-Apt conjugates can be used to directly detect prostate, ovarian, colorectal, and lung cancers or simultaneously detect biomarkers associated with these malignancies. Tenascin-C, mucin 1, prostate-specific antigen, prostate-specific membrane antigen, nucleolin, growth factors, and exosomes are among the cancer biomarkers that can be sensitively detected using such bioconjugates. Furthermore, Apt-conjugated QDs have shown great potential for controlling bacterial infections such as Bacillus thuringiensis, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Campylobacter jejuni, Staphylococcus aureus, and Salmonella typhimurium. This comprehensive review discusses recent advancements in the design of QD-Apt bioconjugates and their applications in cancer and bacterial theranostics.     

On the design of composite protein-quantum dot biomaterials via self-assembly

Incorporation of nanoparticles during the hierarchical self-assembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein-Ultrabithorax (EGFP-Ubx) fusion protein and luminescent CdSe-ZnS core-shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein-nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.     

Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption

Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.     

Binding of human normal and multiple sclerosis-derived myelin basic protein to phospholipid vesicles: effects on membrane head group and bilayer regions

The detailed interaction of human myelin basic protein (MBP) with charged lipids may be critical in organizing the myelin sheath into its biologically functional structure. Carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy has been used to study this interaction by examining spectral consequences of additions of MBP to membrane preparations of the negatively charged lipid phosphatidylglycerol (PG). Lipid head group 13C and 31P linewidths were found to narrow upon addition of protein, while concomitant broadening was noted for bilayer carbon resonances. At intermediate MBP/PG ratios, two components in slow exchange on the NMR time scale (bulk PG and a protein-induced PG domain) were observed for the 13C resonance of the head group carbon atom adjacent to phosphate. These results, and other spectral evidence, suggested that head groups in free PG vesicles are motionally restricted by intermolecular interactions which are disrupted by competition with MBP Lys and Arg positively charged side chains. Titration of PG with the homopolypeptide poly-L-lysine produced comparable effects on PG 13C head group spectra, indicating that electrostatic attractions constitute the primary basis of the observed interactions. Vicinal and/or geminal 13C-31P coupling constants measured from the spectra of PG head group carbons were found to be essentially invariant for free PG in dimethyl sulfoxide solution, free PG vesicles, PG vesicles + MBP, and PG vesicles + poly-L-lysine. Comparison of the spectral effects induced in PG head group resonances by normal vs multiple sclerosis-derived MBP (MS-MBP) indicated that the MS-MBP is relatively less effective in converting PG to the protein-induced domain, a result which was attributed to increased protein self-aggregation arising from the reduced net positive character of the MS protein samples.     

Lead sulfide near-infrared quantum dot bioconjugates for targeted molecular imaging

In this paper, we report the use of lead sulfide quantum dot (PbS QD) bioconjugates as near infrared (NIR) contrast agents for targeted molecular imaging with expanded emission wavelengths beyond 1000 nm. The red-shifted emission band, coupled with the small particle size, which will facilitate clearance, both afford PbS QDs unique properties for noninvasive, high resolution in vivo NIR imaging applications. We have performed imaging experiments at the molecular level using surface-modified PbS NIR QDs, together with our lab-built NIR imaging system. This novel instrumentation and fluorescent contrast agent have enabled us to study the relatively unexplored NIR biomedical imaging spectral region of 900-1200 nm. Preliminary experimental results indicate that PbS-QD/antibody bioconjugates are promising candidates for targeted NIR molecular imaging and future in vivo NIR tissue imaging applications.     

Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).     

Quantitative characterization of quantum dot‐labeled lambda phage for Escherichia coli detection

We characterize CdSe/ZnS quantum dot (QD) binding to genetically modified bacteriophage as a model for bacterial detection. Interactions among QDs, lambda (λ) phage, and Escherichia coli are examined by several cross‐validated methods. Flow and image‐based cytometry clarify fluorescent labeling of bacteria, with image‐based cytometry additionally reporting the number of decorated phage bound to cells. Transmission electron microscopy, image‐based cytometry, and electrospray differential mobility analysis allow quantization of QDs attached to each phage (4–17 QDs) and show that λ phage used in this study exhibits enhanced QD binding to the capsid by nearly a factor of four compared to bacteriophage T7. Additionally, the characterization methodology presented can be applied to the quantitative characterization of other fluorescent nanocrystal‐biological conjugates. Biotechnol. Bioeng. 2009;104: 1059–1067. Published 2009 Wiley Periodicals, Inc.     

Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria

CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.     

Bioconjugated quantum dots for cancer research: Present status,prospects and remaining issues

Semiconductor quantum dots (QDs) are nanoparticles in which charge carriers are three dimensionally confined or quantum confined. The quantum confinement provides size-tunable absorption bands and emission color to QDs. Also, the photoluminescence (PL) of QDs is exceptionally bright and stable, making them potential candidates for biomedical imaging and therapeutic interventions. Although fluorescence imaging and photodynamic therapy (PDT) of cancer have many advantages over imaging using ionizing radiations and chemo and radiation therapies, advancement of PDT is limited due to the poor availability of photostable and NIR fluorophores and photosensitizing (PS) drugs. With the introduction of biocompatible and NIR QDs, fluorescence imaging and PDT of cancer have received new dimensions and drive. In this review, we summarize the prospects of QDs for imaging and PDT of cancer. Specifically, synthesis of visible and NIR QDs, targeting cancer cells with QDs, in vitro and in vivo cancer imaging, multimodality, preparation of QD-PS conjugates and their energy transfer, photosensitized production of reactive oxygen intermediates (ROI), and the prospects and remaining issues in the advancement of QD probes for imaging and PDT of cancer are summarized.     

Synthesis and photophysical studies of CdTe quantum dot-monosubstituted zinc phthalocyanine conjugates

The linkage of unsymmetrically monosubstituted 4-aminophenoxy zinc phthalocyanine (ZnAPPc, 5) to CdTe quantum dots capped with mercaptopropionic acid (MPA), l-cysteine (l-cys) or thioglycolic acid (TGA) has been achieved using the coupling agents ethyl-N(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide, which facilitate formation of an amide bond to form the QD-ZnAPPc-linked conjugate. The formation of the amide bond was confirmed using Raman and IR spectroscopies. Atomic force microscopy (AFM) and UV-Vis spectroscopy were used further to characterise the conjugate. Förster resonance energy transfer (FRET) resulted in stimulated emission of ZnAPPc in both the linked (QD-ZnAPPc-linked) and mixed (QD:ZnAPPc-mixed) conjugates. The linked l-cys and TGA QDs conjugates (QD-ZnAPPc-linked) gave the largest FRET efficiencies hence showing the advantages of covalent linking. Fluorescence quantum yields of QDs were decreased in QD:ZnAPPc-mixed and QD:ZnAPPc-linked.     

Effect of ligand density on the spectral, physical, and biological characteristics of CdSe/ZnS quantum dots

Chemical modification of the surface of CdSe/ZnS quantum dots (QDs) with small molecules or functional ligands often alters the characteristics of these particles. For instance, dopamine conjugation quenches the fluorescence of the QDs, which is a property that can be exploited for sensing applications if the conjugates are taken up into living cells. However, different sizes and/or preparations of mercaptocarboxylic acid solubilized QDs show very different properties when incubated with cells. It is unknown what physical parameters determine a QDs ability to interact with a cell surface, be endocytosed, escape from endosomes, and/or enter the nucleus. In this study, we examine the surface chemistry of QD-dopamine conjugates and present an optimized method for tracking the attachment of small biomolecules to the surface. It is found that the fluorescence intensity, surface charge, colloidal stability, and biological interactions of the QDs vary as a function of the density of dopamine on the surface. Successful targeting of QD-dopamine to dopamine receptor positive PC12 cells correlates with greater homogeneity of particle thiol layer, and a minimum number of ligands required for specific association can be estimated. These results will enable users to develop methods for screening QD conjugates for biological activity before proceeding to experiments with cell lines and animals.     

Uptake of CdSe and CdSe/ZnS quantum dots into bacteria via purine-dependent mechanisms

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.     

Biotin-4-fluorescein based fluorescence quenching assay for determination of biotin binding capacity of streptavidin conjugated quantum dots

The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD-biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

Uptake of CdSe and CdSe/ZnS Quantum Dots into Bacteria via Purine-Dependent Mechanisms

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.     

Comprehensive study of interaction between biocompatible PEG‐InP/ZnS QDs and bovine serum albumin

Polyethylene glycol (PEG) surface modified biocompatible InP/ZnS quantum dots (QDs) act as a potential alternative for conventional carcinogenic cadmium‐based quantum dots for in vivo and in vitro studies. Comprehensively, we studied the interaction between a model protein bovine serum albumin (BSA) and PEGylated toxic free InP/ZnS QDs using various spectroscopic tools such as absorption, fluorescence quenching, time resolved and synchronous fluorescence spectroscopic measurements. These studies principally show that tryptophan (Trp) residues of BSA have preferable binding affinity towards PEG‐InP/ZnS QDs surface and a blue shift in Trp fluorescence emission is a signature of conformational changes in its hydrophobic microenvironment. Photoluminescence (PL) intensity of Trp is quenched by ground state complex formation (static quenching) at room temperature. However, InP/ZnS@BSA conjugates become unstable with increasing temperature and PL intensity of Trp is quenched via dynamic quenching by PEG‐InP/ZnS QDs. Experimentally determined thermodynamic parameters for these conjugates have shown spontaneity, entropy driven and exothermic nature of bio‐conjugation. The calculated binding affinity (n ? 1, Hill coefficient) suggest that the affinity of InP/ZnS QDs for a BSA protein is not dependent on whether or not other BSA proteins are already bound to the QD surface. Energy transfer efficiency (E), Trp residue to InP/ZnS QDs distances and energy transfer rate (k_T) were all obtained from FÖrster resonance energy.