Single-step bioassays in serum and whole blood with a smartphone, quantum dots and paper-in-PDMS chips.

The development of nanoparticle-based bioassays is an active and promising area of research, where point-of-care (POC) diagnostics are one of many prospective applications. Unfortunately, the majority of nanoparticle-based assays that have been developed to date have failed to address two important considerations for POC applications: use of instrumentation amenable to POC settings, and measurement of analytes in biological sample matrices such as serum and whole blood. To address these considerations, we present design criteria and demonstrate proof-of-concept for a semiconductor quantum dot (QD)-based assay format that utilizes smartphone readout for the single-step, Förster resonance energy transfer (FRET)-based detection of hydrolase activity in serum and whole blood, using thrombin as a model analyte. Important design criteria for assay development included (i) the size and emission wavelength of the QDs, which had to balance brightness for smartphone imaging, optical transmission through blood samples, and FRET efficiency for signaling; (ii) the wavelength of a light-emitting diode (LED) excitation source, which had to balance transmission through blood and the efficiency of excitation of QDs; and (iii) the use of an array of paper-in-polydimethylsiloxane (PDMS)-on-glass sample chips to reproducibly limit the optical path length through blood to ca. 250 μm and permit multiplexing. Ultimately, CdSe/CdS/ZnS QDs with peak emission at 630 nm were conjugated with Alexa Fluor 647-labeled peptide substrates for thrombin and immobilized on paper test strips inside the sample cells. This FRET system was sensitive to thrombin activity, where the recovery of QD emission with hydrolytic loss of FRET permitted kinetic assays in buffer, serum and whole blood. Quantitative results were obtained in less than 30 min with a limit of detection 18 NIH units mL(-1) of activity in 12 μL of whole blood. Proof-of-concept for a competitive binding assay was also demonstrated with the same platform. Overall, this work demonstrates that the integration of QDs with smartphones and other consumer electronics can potentiate bioassays that are highly amenable to future point-of-care diagnostic applications.

[1]  Z. Du,et al.  Multiplexed immunoassay biosensor for the detection of serum biomarkers — β-HCG and AFP of Down Syndrome based on photoluminescent water-soluble CdSe/ZnS quantum dots , 2013 .

[2]  Igor L. Medintz,et al.  Emerging non-traditional Förster resonance energy transfer configurations with semiconductor quantum dots: Investigations and applications , 2014 .

[3]  Xiaogang Peng,et al.  Formation of high-quality CdS and other II-VI semiconductor nanocrystals in noncoordinating solvents: tunable reactivity of monomers. , 2002, Angewandte Chemie.

[4]  A. Ozcan,et al.  Quantum dot enabled detection of Escherichia coli using a cell-phone. , 2012, The Analyst.

[5]  Niko Hildebrandt,et al.  Nanobodies and nanocrystals: highly sensitive quantum dot-based homogeneous FRET immunoassay for serum-based EGFR detection. , 2014, Small.

[6]  W. Russ Algar,et al.  Luminescent terbium complexes: Superior Förster resonance energy transfer donors for flexible and sensitive multiplexed biosensing , 2014 .

[7]  Eleonora Petryayeva,et al.  Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots. , 2014, Analytical chemistry.

[8]  Henry Pinkard,et al.  Advanced methods of microscope control using μManager software. , 2014, Journal of biological methods.

[9]  Zongwen Jin,et al.  Semiconductor quantum dots for in vitro diagnostics and cellular imaging. , 2012, Trends in biotechnology.

[10]  Jerry C. Chang,et al.  Biocompatible quantum dots for biological applications. , 2011, Chemistry & biology.

[11]  G. Whitesides,et al.  Diagnostics for the developing world: microfluidic paper-based analytical devices. , 2010, Analytical chemistry.

[12]  Yingfu Li,et al.  Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling. , 2004, Chemistry.

[13]  Derek Tseng,et al.  Fluorescent imaging of single nanoparticles and viruses on a smart phone. , 2013, ACS nano.

[14]  Hongying Zhu,et al.  Optical imaging techniques for point-of-care diagnostics. , 2013, Lab on a chip.

[15]  Igor L. Medintz,et al.  Quantum Dots in Bioanalysis: A Review of Applications across Various Platforms for Fluorescence Spectroscopy and Imaging , 2013, Applied spectroscopy.

[16]  Derek K. Tseng,et al.  Imaging and sizing of single DNA molecules on a mobile phone. , 2014, ACS nano.

[17]  J. Frangioni,et al.  Image-Guided Surgery Using Invisible Near-Infrared Light: Fundamentals of Clinical Translation , 2010, Molecular imaging.

[18]  David K. Wood,et al.  Nanoparticles That Sense Thrombin Activity As Synthetic Urinary Biomarkers of Thrombosis , 2013, ACS nano.

[19]  W. Zijlstra,et al.  Absorption spectra of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin. , 1991, Clinical chemistry.

[20]  U. Krull,et al.  Quantum dot and gold nanoparticle immobilization for biosensing applications using multidentate imidazole surface ligands. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[21]  Hojeong Yu,et al.  Smartphone fluorescence spectroscopy. , 2014, Analytical chemistry.

[22]  Matthew B. Johnson,et al.  Large-scale synthesis of nearly monodisperse CdSe/CdS core/shell nanocrystals using air-stable reagents via successive ion layer adsorption and reaction. , 2003, Journal of the American Chemical Society.

[23]  A. Roda,et al.  Integrating biochemiluminescence detection on smartphones: mobile chemistry platform for point-of-need analysis. , 2014, Analytical chemistry.

[24]  Annalisa D'Andrea,et al.  Lateral flow assay with near-infrared dye for multiplex detection. , 2013, Clinical chemistry.

[25]  Hedi Mattoussi,et al.  Luminescent quantum dots as platforms for probing in vitro and in vivo biological processes. , 2012, Advanced drug delivery reviews.

[26]  Ulrich J Krull,et al.  Beyond labels: a review of the application of quantum dots as integrated components of assays, bioprobes, and biosensors utilizing optical transduction. , 2010, Analytica chimica acta.

[27]  Guodong Liu,et al.  Aptamer-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for protein analysis. , 2009, Analytical chemistry.

[28]  Jesse V Jokerst,et al.  Nano-bio-chips for high performance multiplexed protein detection: determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels. , 2009, Biosensors & bioelectronics.

[29]  P. Yager,et al.  Point-of-care diagnostics for global health. , 2008, Annual review of biomedical engineering.

[30]  Yi Lin,et al.  On-chip dual detection of cancer biomarkers directly in serum based on self-assembled magnetic bead patterns and quantum dots. , 2013, Biosensors & bioelectronics.

[31]  References , 1971 .

[32]  V. Biju,et al.  Delivering quantum dots to cells: bioconjugated quantum dots for targeted and nonspecific extracellular and intracellular imaging. , 2010, Chemical Society reviews.

[33]  Eleonora Petryayeva,et al.  Proteolytic assays on quantum-dot-modified paper substrates using simple optical readout platforms. , 2013, Analytical chemistry.

[34]  Igor L. Medintz,et al.  Synthesizing and modifying peptides for chemoselective ligation and assembly into quantum dot-peptide bioconjugates. , 2013, Methods in molecular biology.

[35]  Igor L. Medintz,et al.  Solution-phase single quantum dot fluorescence resonance energy transfer. , 2006, Journal of the American Chemical Society.

[36]  X. Le,et al.  Aptamer binding assays for proteins: the thrombin example--a review. , 2014, Analytica chimica acta.

[37]  C. Fan,et al.  Ultrasensitive, multiplexed detection of cancer biomarkers directly in serum by using a quantum dot-based microfluidic protein chip. , 2010, ACS nano.

[38]  Subinoy Rana,et al.  Nanoparticles for detection and diagnosis. , 2010, Advanced drug delivery reviews.

[39]  Niko Hildebrandt,et al.  Quantum-dot-basedFörster resonance energy transfer immunoassay for sensitive clinical diagnostics of low-volume serum samples. , 2013, ACS nano.

[40]  Xiaohu Gao,et al.  Designing multifunctional quantum dots for bioimaging, detection, and drug delivery. , 2010, Chemical Society reviews.

[41]  M. Lancé.  A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis , 2015, Thrombosis Journal.

[42]  Itamar Willner,et al.  Optical molecular sensing with semiconductor quantum dots (QDs). , 2012, Chemical Society reviews.

[43]  Erlong Zhang,et al.  A review of NIR dyes in cancer targeting and imaging. , 2011, Biomaterials.

[44]  S. Coughlin,et al.  Thrombin signalling and protease-activated receptors , 2000, Nature.

[45]  Igor L. Medintz,et al.  Active cellular sensing with quantum dots: transitioning from research tool to reality; a review. , 2012, Analytica chimica acta.