Article pubs.acs.org/ac Quantum Dot/Carrier−Protein/Haptens Conjugate as a Detection Nanobioprobe for FRET-Based Immunoassay of Small Analytes with All-Fiber Microfluidic Biosensing Platform † ‡ † § † Feng Long, , Chunmei Gu, April Z. Gu, and Hanchang Shi*, † State Key Joint Laboratory of ESPC, School of Environment, Tsinghua University, Beijing 100084, People’s Republic of China ‡ Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § Department of Civil and Environmental Engineering, Northeastern University, Boston, Massachusetts 02115, United States *S Supporting Information ABSTRACT: This study demonstrates the use of carrier- protein/haptens conjugate (e.g., BSA/2,4-dichlorophenoxy- acetic acid, 2,4-D-BSA) for biological modification of quantum dots (QDs) for the detection of small analytes. Bioconjugated QDs, which are used as a detection nanoimmunoprobe, were prepared through conjugating carboxyl QDs with 2,4-D-BSA conjugate. Based on the principle of quantum dot− fluorescence resonance energy transfer (QD-FRET), an all- fiber microfluidic biosensing platform has been developed for investigating FRET efficiency, immunoassay mechanism and format, and binding kinetics between QD immunoprobe and fluorescence labeled anti-2,4-D monoclonal antibody. The structure of multiplex-haptens/BSA conjugate coupling to QD greatly improves the FRET efficiency and the sensitivity of the nanosensor. With a competitive detection mode, samples containing different concentrations of 2,4-D were incubated with a given concentration of QD immunoprobe and fluorescence-labeled antibody, and then detected by the all-fiber microfluidic biosensing platform. A higher concentration of 2,4-D led to less fluorescence-labeled anti-2,4-D antibody bound to the QD immunoprobe surface and, thus, a lower fluorescence signal. The quantification of 2,4-D over concentration ranges from 0.5 nM to 3 μM with a detection limit determined as 0.5 nM. The performance of the nanosensor with spiked real water samples showed good recovery, precision, and accuracy, indicating that it was less suspectable to water matrix effects. With the use of different QD nanobioprobes modified by other carrier-protein/ haptens conjugates, this biosensing protocol based on QD-FRET can be potentially applied for on-site, real-time, inexpensive, and easy-to-use monitoring of other trace analytes. ost of the analytes of environmental interest, such as Nanometer-sized quantum dots (QDs) have emerged as M pesticides, persistent organic pollutants (POPs), endo- promising alternative bioanalytical tools, because of their crine-disrupting chemicals (EDCs), explosives, and toxins, unique optical properties, including high quantum yield, possess a molecular weight of <1 kDa, and they cause many photostability, narrow emission spectrum, and broad absorp- − 5,6 severe health problems at very low concentrations.1 3 Until tion. The narrow, size-tuned, and symmetric emission spectra fl recently, the quantification of small analytes has been limited to of QDs have made them excellent donors for uorescence the traditional chromatographic and spectroscopic technolo- resonance energy transfer (FRET) sensors, and they greatly reduce the overlap between the emission spectra of donor and gies. These methods, although accurate with low detection acceptor and circumvents the cross-talk in such FRET pairs.5,6 limits, are labor-intensive and require expensive and sophisti- Meanwhile, QDs have broad excitation spectra as donors, and cated instrumentation, as well as complicated and multistep they allow excitation at a single wavelength far removed (>100 sample preparation, which prohibits potential real-time and on- nm) from their respective emissions, which can avoid the direct site practical applications. Therefore, considerable research excitation of the acceptor. In addition, the high photobleaching interests have risen for detecting low levels of small molecules threshold and good chemical stability of QDs greatly improve in biosensor developments. Among them, nanoparticle (NP)- the detection sensitivities and detection limits.6,7 Therefore, based biosensors have gained great attention, because of their QD-based FRET biosensors have been widely used in simplicity, robustness, sensitivity, specificity, and cost-effective- immunoassay,7,8 medical imaging,9,10 clinical/diagnostic as- ness.4 NP-based biosensors can usually carry out analyte recognition and binding, as well as convert the biorecognition Received: January 5, 2012 event to a measurable optical or electric signal through an Accepted: March 20, 2012 integrated signal transduction system.4 Published: March 20, 2012 © 2012 American Chemical Society 3646 dx.doi.org/10.1021/ac3000495 | Anal. Chem. 2012, 84, 3646−3653 Analytical Chemistry Article Figure 1. Preparation of QD/carrier−protein/haptens nanoimmunoprobe and immunoassay mechanism for the detection of 2,4-D: (a) synthesis of 2,4-D-BSA conjugates and (b) synthesis of QD/2,4-D-BSA immunoprobe. (c) Depiction of the competitive immunoassay mechanism of 2,4-D based on QD-FRET. says,11 and biomolecular binding assay.12 Antibody bioconju- ■ EXPERIMENTAL SECTION gates of QDs, prepared using covalent or noncovalent linking Materials. Bovine serum albumin (BSA), 2,4-dichlorophe- approaches, are the most developed and widespread detection noxyacetic acid (2,4-D), 2,4-dichlorophenol (2,4-DCP), 4-(2,4- 7,8,13 bioprobes to integrating QDs into bioanalyses. However, dichlorophenoxy)butyric acid (2,4-DB), 2,4,5-trichlorophe- the control over the number of antibodies per QD and their noxyacetic acid (2,4,5-T), 4-chlorophenoxyacetic acid (CPA), orientation and position relative to the QD is very difficult. 4-chloro-o-tolyloxyacetic acid (MCPA), 1-ethyl-3-(3-dimethy- Because of the possibility of inadvertently disrupting the lamino-propyl) carbodiimide hydrochloride (EDC), N,N′- binding site when conjugating QD with antibody, the activity dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide − loss of antibody is inevitable.6,11,14 In addition, antibodies (NHS) were purchased from Sigma Aldrich (Steinheim, usually must be cryopreserved, but QDs cannot be frozen, Germany). All solutions were prepared with ultrapure water from a Millipore Milli-Q sytem. All the other reagents, unless which makes storage of the QD antibody a major obstacle for specified, were supplied by the Beijing Chemical Agents its practical applications. (China). All chemicals were of analytical reagent (AR) grade. ff To e ectively address these challenges, we have developed Qdot 605 ITK carboxyl quantum dots were obtained from carrier−protein−haptens-coupled QD nanobioprobe protocols Invitrogen, Ltd. (USA). Transmission electron microscopy to perform rapid and sensitive detection of small targets in real (TEM) (Model H-7650B, Hitachi, Japan) images revealed that water samples. 2,4-Dichlorophenoxyacetic acid (2,4-D, MW = the Qdots were uniform in size, and only the inorganic particles 221.04), one of the most widely used pesticides worldwide,15 are directly visualized at ∼10 nm × 5 nm (see Figure S-1a in was selected as a model target. 2,4-D has been shown to lead to the Supporting Information). The size of the polymer-capped ∼ − cancer in humans,16 endocrine-disrupting activities,15,17 and QDs ( 9 15 nm) was also characterized by dynamic laser degenerative changes in the nervous system.18 QD nano- scattering (DLS) (see Figure S-1b in the Supporting Information) immunoprobes were prepared by conjugating carboxyl QDs Dye Cy5.5 labeled monoclonal anti-2,4-D antibody (Refer- with a 2,4-D-BSA conjugate, which is regarded as the ence No. 2H8) were produced by our research group.19 The immunological recognition of the anti-2,4-D antibody, as well affinity and specificity of the produced monoclonal antibodies as being used for optical transduction. To improve upon to 2,4-D were characterized in conjugate-coated ELISA formats. existing immunoassay techniques (e.g., ELISA, SPR, and The hapten−carrier conjugate 2,4-D-BSA was prepared by microarray) based on static solid/liquid interface reaction, conjugating an amine of BSA to 2,4-D through the DCC/NHS regarding their sensitivity, overall analysis time, and simplified chemistry, which is described as follows (Figure 1a). 2,4-D manipulation, an all-fiber microfluidic biosensing platform has (22.0 mg) and NHS (11.5 mg) were dissolved in 2 mL of been developed. The ambulatory fluid is expected to accelerate dimethyl formamide (DMF), and then activated by dropwise the reaction among analytes, the fluorescence-labeled antibody, adding 20.6 mg of DCC. The mixture was stirred at room temperature for 18 h and then centrifugated at 5000 rpm. The and the QD-BSA-2,4-D nanoconjugate, as well as prevent QDs fi supernatant was added drop by drop to create a 30-mL volume from nonspeci c deposition at surfaces. The binding kinetics of of 5 mg/mL BSA solution for conjugation. The reaction fl the QD-BSA-2,4-D immunoprobe and the uorescence-labeled mixture was then dialyzed at 4 °C and lyophilized to get the anti-2,4-D monoclonal antibody, and the sensitivity and conjugates. The estimated number of hapten molecules selectivity of this nanoimmunosensor, based on QD-FRET, attached to the carrier protein (hapten to protein molar ratio, have also been evaluated. MR) BSA, determined by matrix-assisted laser desorption 3647 dx.doi.org/10.1021/ac3000495 | Anal. Chem. 2012, 84, 3646−3653 Analytical Chemistry Article ionization−time of flight (MALDI-TOF) mass spectroscopy end of a multimode fiber, which directly