Decoding of Quantum Dots Encoded Microbeads Using a

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Decoding of Quantum Dots Encoded Microbeads Using a Hyperspectral Fluorescence Imaging Method Yixi Liu,†,‡ Le Liu,§ Yonghong He,*,†,‡ Liang Zhu,†,‡ and Hui Ma†,‡ †

Shenzhen Key Laboratory for Minimal Invasive Medical Technologies, Institute of Optical Imaging and Sensing, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China ‡ Department of Physics, Tsinghua University, Beijing 100084, China § Institute of Green Chemistry and Energy, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China ABSTRACT: We presented a decoding method of quantum dots encoded microbeads with its fluorescence spectra using line scan hyperspectral fluorescence imaging (HFI) method. A HFI method was developed to attain both the spectra of fluorescence signal and the spatial information of the encoded microbeads. A decoding scheme was adopted to decode the spectra of multicolor microbeads acquired by the HFI system. Comparison experiments between the HFI system and the flow cytometer were conducted. The results showed that the HFI system has higher spectrum resolution; thus, more channels in spectral dimension can be used. The HFI system detection and decoding experiment with the single-stranded DNA (ssDNA) immobilized multicolor beads was done, and the result showed the efficiency of the HFI system. Surface modification of the microbeads by use of the polydopamine was characterized by the scanning electron microscopy and ssDNA immobilization was characterized by the laser confocal microscope. These results indicate that the designed HFI system can be applied to practical biological and medical applications.

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conventional barcoding material. The photoluminescent gold nanoparticles have low toxicity and facile synthesis under ecofriendly conditions,13 but their stability can be an issue when under the continuous excitation with UV/blue light.14 The photonic crystals, using their characteristic reflection peaks as the encoding element, can be very stable and free of the bleaching and quenching problem. Great improvement has been made to get a great number of distinguishable codes by the use of photonic crystals.15 However, their incorporating photonic crystals into the patterned graphics way has stringent orientation requirement, which limited the decoding efficiency of the system. Besides, structural colors of photonic crystals which are derived from Bragg reflection are angle dependent, adding another complexity for the decoding process.16 The upconversion nanoparticles, which can emit visible light under the infrared irradiation, have more merits: low autofluorescence from biological samples, which can achieve high signal-to-noise ratio; less scattering and absorption, which can enhance the quality of the image; and deep penetration in biological tissues.17 These merits show a great promise of upconversion nanoparticles for biological applications. Recently, anticounterfeiting application has been reported by Zhang et al.18 by use of the upconversion microrods. But, the most serious limitation

ncoded microspheres are an important part for a multiplexed assay, which can simultaneously measure multiple analytes at one time. Various encoding strategies can be used to encode microspheres such as optical, chemical, graphical, electronic, and physical encoding strategy.1 Among them, the optical encoding is the most widely used strategy due to its simplicity, rapid readout, and compatibility with a variety of biological chemistries.2 The major feature of optical encoding of microbeads is utilizing the uniqueness of the emission spectra profile of the fluorescent labeling element, which is carried by the microbeads. In the past two decades, quantum dots (QDs, also called semiconductor nanocrystals) have been paid more and more attention due to their extensive applications such as cytotoxicity screening, genetic analysis, and protein detection.3−8 They have gradually replaced the conventional labeling element (e.g., organic dyes) to be a more ideal fluorescent label9 due to their excellent optical properties such as broad absorption spectrum and narrow and symmetric emission spectrum. Compared with organic dyes, QDs are 10−20× brighter10 and have less photobleaching effect.11 Also, the emission peak of QDs is tunable by controlling its size, which facilitates the integration of QDs into large-scale multiplexed assays.12 Aside from the QDs, there are many newly emerged labeling materials, for example, gold nanoparticles, photonic crystals, and upconversion nanoparticles (UCNPs). All these materials have their unique features and are taken as the promising alternatives for the © XXXX American Chemical Society

Received: January 30, 2015 Accepted: April 22, 2015

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DOI: 10.1021/acs.analchem.5b00398 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Schematic illustration of (a) the HFI system and (b) the spectrometer: laser light, LS; collimation lens, L1; cylindrical convex lens, CL; dichroic mirror, DM; achromatic lens, L2; long pass filter, F; spectrometer, S; detector, D.

single one. Besides, in a spectrofluorometer, the data in a spectrum are not recorded simultaneously and may be affected by the time-dependent noise such as the variation of the light source. The increasing need for simultaneous identification of multiple analytes (e.g., DNA, antibody, enzyme) in biological, combinatorial chemistry, and clinical diagnostics applications42 is the driving force for researchers to exploit the new coding dimensions.43−46 Recently, great improvements have been made by Jin’s group45,47 that they create a temporal coding dimension in a wide microsecond-to-millisecond range by control the fluorescence lifetime of UCNPs. In this paper, we presented a decoding method of QD-labeled microbeads with its fluorescence spectra using line scan hyperspectral fluorescence imaging (HFI) method.48 Both the spectra and the spatial information of the encoded microbeads can be acquired with this method. The spectra of several beads that are on the line of illumination can be obtained at the same time, which means an additional spatial dimension is created. Highly overlapping spectra can be separated with the combination of the HFI method and the decoding scheme. A performance comparison experiment between the HFI system and the flow cytometer was conducted, and the experimental results showed that the HFI system has higher spectrum resolution and can obtain both the spectra of fluorescence signal and the spatial information of the encoded microbeads. Higher spectrum resolution means better ability to separate spectra code with a slight difference and the possibility for more analytes to be encoded. The spatial information, which the HFI system attained and other methods do not have, provides an additional dimension to this system and gives the method a high detection throughput. Finally, the signal of a real sample that deposits the polydopamine on the surface of the multicolor microbeads and immobilize the ssDNA onto it was decoded using the HFI system.

for the upconversion nanoparticles is the low quantum yield, which varies from 0.005−0.3%.19 Recently, through homogeneous doping of the UCNPs, the quantum yield can be as high as 0.89 ± 0.05%.20 However, compared with the quantum yield of the QDs, which can be 30−86%,21−24 the quantum yield of UCNPs is still very low. Encoding Part. The principle of encoding25 that incorporates QDs into microspheres is combining the different wavelength emitted by the different color of QDs with different intensity levels of each color, and the encoding element is the spectral signature of the encoded beads. Theoretically, six-color QDs with 10 intensity level can create a code library with the number of about one million. Then the desired analytes can be coupled onto the surface of encoded microbeads for identification. There are mainly three basic ways to incorporate QDs into microbeads: layer-by-layer assembly,26,27 embedding QDs into microbeads during synthesis,28 and the swelling method, which was first proposed by Nie’s group.25 The swelling method is the most widely used method, and many other approaches have been proposed based on it.29,30 To ensure all the microbeads are perfectly uniform is a great challenge, and the small variations in bead size will cause the fluctuation of the absolute intensity between encoded beads.31 Thus, the ratiometric way is chosen to encode signal instead of absolute intensity-based way. Decoding Part. Generally, intensity information of QDlabeled microbeads can be recorded using flow cytometer,32−35 which indeed helps achieve a rapid readout, multiplexed detection system. However, it is a filter-based instrument; thus, some limitations need to be considered:32 (1) the emission wavelength of QDs should be within the detection channels of the flow cytometer, which may lower the degree of multiplexing; (2) preventing the situation of simultaneous detection by two channels, there exists a threshold between emission peaks of two labeling QDs. Another approach to record the signal of labeled microbeads is to use a spectrofluorometer,4,36−41 which can record the spectrum profile instead of fluorescence intensities of the encoded microbeads. Nevertheless, the spectrofluorometer can only record the fluorescence signal of ensemble QDs instead of a

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MATERIALS AND METHODS Optical System. Figure 1a shows the optical layout of the line scan HFI system. The laser light (LS, 405 nm, 50 mw) was collimated by a collimation lens (L1, f = 25.4 mm) and passed a B


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Analytical Chemistry cylindrical convex lens (CL, f = 25 mm). The beam was then reflected by a dichroic mirror (DM, reflection band: 350−475 nm) and focused by an achromatic lens (L2, f = 30 mm) to form a line illumination on the sample. The fluorescence emitted by the sample was collected by the same achromatic doublet (L2) and transmitted through the dichroic mirror with the transmission band of 492−950 nm, which can cover most fluorescent wavelengths of QDs. After the dichroic mirror, the light passes through the long pass filter (F) with the cut-on wavelength of 500 nm to block the reflected laser light. Following the filter, there was a spectrometer (S) to separate the different wavelengths of the fluorescence and a detector (D) to record the data. The home-built spectrometer in Figure 1a consists of a transmission grating (25 × 25 mm2, 300 lines/mm) and an achromatic lens (L3, f = 200 mm), as shown in Figure 1b. The fluorescence exited the transmission grating with a different angle corresponding to their wavelength and then focused on the detector by an achromatic lens. The detector has 3352 × 2532 pixels with each pixel size of 5.4 × 5.4 μm2. The spectrometer was designed with the center wavelength of 560 nm and can acquire data over a spectral range of 300 nm (from 410 to 710 nm, with the resolution of 4 nm). Considering the emission wavelength of most commercially available QDs are between 450 and ∼660 nm, it can totally meet the requirement of a multiplexing system. The resolution can be adjusted by changing the focus length of the lens and density of the grating. The simple illustration of how this system works with recorded data is shown in Figure 2. We encode two types of microbeads:

photoluminescence (PL). All measurements were performed at room temperature under ambient conditions. Characterization of Quantum Dots. ZnS-capped CdSe QDs (Wuhan Jiayuan Quantum Dot Technological Development Corporation) are used in this paper. The emission peaks of four kinds of QDs are 519, 525, 585, and 619 nm, respectively. The full width at half-maximum (fwhm) of these QDs are 45∼50 nm. Figure3 shows the emission spectra of quantum dots, which were recorded by a spectrofluorometer (FL-1039/40, Horiba).

Figure 3. Four emission lines corresponding to four different types of QDs with wavelengths of 519, 525, 585, and 619 nm.

Characterization of Microspheres. The diameter of highly cross-linked monodisperse poly(chloromethylstyreneco-divinylbenzene) microspheres (provided by Nano-Micro Research Center, Peking University) is approximately 10 μm, as shown in Figure 4. The size and morphology of microbeads

Figure 4. Scanning electron microscope images of highly cross-linked poly(chloromethylstyrene-co-divinylbenzene) microspheres with the magnification of (a) 1 and (b) 20k.

Figure 2. (a) Schematic illustrations of two encoded microbeads excited by the line laser. (b) The original image of fluorescence captured by the CCD detector. (c) Upper and lower side of spectra are from bead1 and bead2 shown in (a), respectively.

were characterized by scanning electron microscope (S4800, Hitachi) operating at 5 kV. Figure 4a was taken with a magnification of 1k times shows the uniformity of the bead size, and Figure 4b, which was taken with a magnification of 20k times, shows the porous features of the surface of cross-linked microspheres. Encapsulation Method. The swelling method proposed by Nie25 is the most popular incorporation method because of its facile and rapid operating process. Two variants of the swelling methods, the conventional swelling method and self-healing encapsulation strategy,30 were adopted to embed QDs into the porous microbeads, and their laser confocal microscope (FV1000, Olympus) images are shown in Figure 5a and b, respectively. A “bright ring” exists near the surface of the microbeads, as shown in Figure 5a, which means the QDs are less protected and easier to quench, leading to the PL decreasing of the encoded beads. Also, the aggregation of the QDs near the bead surface is unfavorable for the analyte

the first type is labeled with QDs with the wavelength of 525 nm (QDs525 nm) and 585 nm (QDs585 nm) with an intensity ratio of 1:1 (code1); the second kind is labeled with only QDs619 nm (code2). Figure 2a schematically shows the line laser excited two encoded microbeads (denoted with color dots), which were deposited onto a glass slide before detection. Figure 2b shows one image captured by the detector, while the laser excites two encoded microbeads as in Figure 2a. With each row representing the spectrum of one certain point in the line area, Figure 2b is the spectrum of the illuminated line area. The marked yellow rows in Figure 2b corresponding to the two microbeads in Figure 2a are plotted in Figure 2c. Upper side and the lower side of Figure 2c are the spectra of the encoded bead with code1 and code2, respectively, after normalizing their C


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reconstruction process in which the low-frequency components are keep untouched. However, the background cannot be completely removed after the symlet wavelets denoising. So, we subtracted the average reflection light of the original blank beads, which have the same concentration with the encoded beads sprayed on the glass slide. After the background subtraction the next step was to unmix the spectra. Here we use the Nelder−Mead algorithm53 to decompose the overlapping signal of the multicolor beads into its component spectra. First, the emission spectrum of single color QD was modeled as the Gaussian curve, which is a·exp{−[(x − b)/c]2}. There are three parameters (a, b, c) that represent the height, peak position, and width of the spectrum, respectively. The signal of beads can be represented as the sum of Gaussian curves with different parameter sets; for example, if two colors of QDs are used, then the signal should be the sum of two Gaussian curves with the parameter sets (a1, b1, c1) and (a2, b2, c2). To implement the algorithm, the first estimation of peak position and width should be given. Then this algorithm will find the component spectra and compare with the original data; if their difference is larger than the predefined error before the implementation, then the algorithm will loop back until the difference is smaller than the error. In this paper, we use Matlab (MathWorks, Inc.) to implement the deconvolving method, and the wavelet denoising process has used the functions from the Matlab wavelet toolbox.

Figure 5. Laser confocal microscope image of encoded microbeads prepared by (a) swelling method and (b) self-healing encapsulation strategy.

immobilization. In contrast, the QDs in Figure 5b were homogeneously located inside the microbeads. So, self-healing encapsulation strategy was used to encapsulate QDs into microbeads in this work. Preparation of Encoded Beads. Typically, 10 mg microbeads were added into 2 mL of the hexadecane and sonicated for 10 min. Then, QDs were dispersed in a mixture solution (25 μL of isopropanol and 475 μL of chloroform) and sonicated for 10 min. The dissolved QDs were then mixed with the resulting suspension of the microbeads and sonicated for 10 min. Then, the resulting mixture was transferred to a three-neck flask in an oil bath and stirred (under the protection of argon) for 1.5 h at a temperature of 50 °C. After this phase, the temperature was slowly raised to 180 °C and remained for 10 min in order to evaporate all the chloroform and isopropanol. Following that, the resulting solution was cooled down rapidly to room temperature. Then the microbeads were collected by centrifuging at 400 G for 5 min and washed with ethanol/ cyclohexane (4:1, v/v) for three times. Finally, the microbeads were sprayed onto the glass slide. Preparation of Polydopamine-Coated Encoded Beads and DNA-QDs Immobilization. The polydopamine-coated encoded beads (PDA@encoded-PSs) were prepared according to the literature,49,50 with some modifications. First, the encoded beads were dispersed in Tris-HCl buffer solution (10 mM, pH = 8.5) and gently stirred for 25 min and collected by centrifuging. Then the encoded beads (0.2 wt %) were redispersed in Tris-HCl buffer, and the dopamine hydrochloride (99%, Alfa Aesar Co., Inc.) was dissolved in the resulting beads-Tris-HCl solution with a concentration of 2 mg/mL. The mixture was stirred for 24 h at 37 °C. The asprepared products were separated by filtration and centrifuged and washed with deionized water for several times. Then, the PDA@encoded-PSs were dispersed in Tris-HCl buffer solution, followed by the addition of DNA-QDs. The mixture was stirred for 6 h at 57 °C. After the immobilization, the solution was centrifuged and washed several times with 0.1 wt % SDS in phosphate buffer solution (prepared by NaH2PO4 and Na2HPO4, pH = 7.4), then dispersed in deionized water. Herein the QD-DNA-PDA@encoded-PSs were obtained and stored at 4 °C for further use. Deconvolving Method. The deconvolving method was based on the algorithm proposed by Lee et al.51 The first step of deconvolving is denoising. First, mean filtering is adopted to remove the salt and pepper noise. Then, wavelet analysis is applied to remove the Gaussian noise and speckle noise. This step mainly consists of two parts: the first part is decomposition of the signal using the symlet wavelet, which is a modified version of Daubechies wavelets with increased symmetry,52 with decomposition level of five; the second part is the

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RESULTS AND DISCUSSION Ideally, if the fwhm of the QDs is very narrow, the spectra can be directly decoded without any deconvolving algorithm. Nonetheless, it is difficult to obtain large-batch produced QDs (commercially available QDs) with stability, high quantum yields, and narrow fwhm.54 Even if the intrinsic fwhm of QDs is very narrow, there are still many factors that may broaden the fwhm of QDs, such as the aggregation of QDs and the rise of the temperature. Besides, for a multiplexing system, overlapping is unavoidable because the possibility of overlapping is highly related to the degree of multiplexing in the spectral domain. Thus, the deconvolving algorithm mentioned above is necessary. Figure 6 illustrates how our deconvolving algorithm works with the experimental data. The microbeads used to demonstrate this process were incorporated with QDs585 nm and QDs619 nm by the ratio of 0.38:1. Raw data acquired by the detector of this encoded bead is shown in Figure 6a. The denoising result is shown in Figure 6b. After the background subtraction (Figure 6c), the unmixed result of the overlapped spectra after the decoding is shown in Figure 6d: red solid line represents the unmixed spectrum of embedded QDs619 nm, the blue solid line represents the unmixed spectrum of embedded QDs585 nm, and the dashed line represents the combined spectrum of them. This decoding scheme can eliminate the stringent requirements for the uniformity of microbeads. We should note that the emission spectra were shifted, and many reasons may account for this: the aggregation of the QDs,55 the interdot energy transfer,56 interaction between QDs and the polymer matrix,57 pressure,58 temperature,55,59,60 and oxidation.61−65 Fukumura et al. have a good summary of the reasons for the spectral shift.66 The thickness of the ZnS layer has the effect on shifting that the QDs will be more resistant to the spectral shifting as the ZnS layer grows thicker. The aggregation of QDs will cause the spectrum red shift due to the Förster resonance energy transfer between different colors of QDs. Some of the effects are size-dependent, D


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Figure 7. (a) Laser confocal microscope image of encoded microbeads using the encapsulation method reported by Riegler et al. (b−d) The decoding results of encoded microbeads with different maximum fluorescence intensities.

Figure 6. Spectra data processing steps. (a) Raw data obtained from the detector; (b) Denoising using signal processing method; (c) Background subtraction; (d) The unmixed overlapping spectra after decoding. Red solid line represents the unmixed spectrum of embedded QDs619 nm, while blue solid line represents the unmixed spectrum of embedded QDs585 nm, and the dashed line represents the combined spectrum of them.

beads were the microbeads encapsulated with QDs519 nm (Color1) and QDs585 nm (Color3). Two batches of encoded microbeads were made, and in each batch, the colors of QDs being used are the same but have different ratios. Due to the difference of temperature and oxidation during the preparation process, their spectra have some differences. The fluorescence signal of these two types of microbeads is detected with the flow cytometer and the HFI system, respectively. The experimental results show that (1) the flow cytometer dots plot of both type-A and type-B beads with two different ratios were overlapped with each other, as shown in Figure 8a and d, respectively; (2) the ratio of these two types of encoded microbeads are different under HFI system. The two different ratios of type-A beads are 0.178:1 (with the variance of 9.9 × 10−4) and 0.195:1 (with the variance of 8.5 × 10−4), as shown in Figure 8b,c. The two different ratios of type-B beads are 0.24:1 (with the variance of 3.6 × 10−4) and 0.32:1 (with the variance of 4.3 × 10−4), as shown in Figure 8e,f. The blue line denotes the decoded spectrum of QDs519 nm in Figure 8b,c and QDs525 nm in Figure 8e,f. The red line denotes the decoded spectrum of QDs585 nm. The combined spectrum is denoted with black dashed line. Thus, the HFI system can identify smaller difference of intensity ratio between colors than the flow cytometry system, which can extend the encoding library. In the following experiment, we made four batches of type-A (solid lines in Figure 9) and type-B beads (dashed lines in Figure 9), and in each batch the intensity ratios of type-A and type-B beads were almost the same (0.55, 0.59, 0.65, and 0.75, respectively). For example, in batch1, the ratio between QDs525 nm and QDs585 nm in type-A beads is 0.556:1 and the ratio between QDs519 nm and QDs585 nm in type-B beads is 0.543:1. In each batch, type-A and type-B beads are unseparable under the flow cytometer due to the intrinsic limitation of the filter-based instrument, which is proven above. Figure 9 shows the decoding results of the HFI system. The solid lines denoted the decoding results of type-A beads and the dashed lines denoted the decoding results of type-B beads. The color of the dashed vertical line denoted the corresponding color of QDs being used and the peak positions of QD585 nm of type-A and type-B beads were purposely aligned in each image of Figure 9. According to the experimental result, the encoded type-A and

for instance, the temperature.67 Besides, the deconvolving algorithm cannot get the exact spectrum as the original one but with the slight variations.51 Among all the effects that may cause spectral shifting, there are two dominate factors:66 temperature and oxidation. The temperature will lead to a red shift of the CdSe QDs, when it is above 100 K,55 due to the dilation of the crystal lattice and lattice-electron interactions. The oxidization will result in a blue shift due to the shrink of the QDs core, and the large blue-shift has been reported in the literature.61,68−70 Again, although the spectral shifting exists, the uniqueness of the spectral signature will not change. To further confirm the effectiveness of this decoding scheme, another encapsulation strategy71 reported by Riegler et al. is adopted to obtain the encoded microbeads labeled with QD525 nm and QD619 nm. This strategy can be seen as another variation of the “swelling” method, except its driving force is hydrophobic interactions. A typical laser confocal microscope image in the observing field is presented in Figure 7a in which fluorescent intensity is not homogeneous between microbeads. The maximum fluorescence intensities and the signal-to-noise ratio (SNR) from Figure 7d to b are decreasing, while decoding results showed the spectra obtained through this encapsulation strategy can still be effectively decoded. As the encoding capacity and decoding scheme are highly related, further enhancement of this decoding method can be made to increase the multiplexing capability. Comparison between the Flow Cytometer and the HFI System. Flow cytometric analysis was performed on BD FACSCalibur flow cytometer. FL1 channel (530/30 nm bandpass filter) detects the green fluorescence (QDs519 nm and QDs525 nm) and FL2 channel (585/42 nm bandpass filter) detects the orange fluorescence (QDs585 nm). The wavelength of excitation laser is 488 nm. In this experiment, three colors of QDs are used to label the microbeads. Type-A beads were the microbeads encapsulated with QDs525 nm (Color2) and QDs585 nm (Color3), while type-B E


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Figure 8. (a) Merging image of upper and lower inset. Upper inset (blue) and lower inset (red) are flow cytometer dots plot of type-A beads with two different ratios. (b) The HFI system decoded spectra with the ratio of 0.178:1. (c) The HFI system decoded spectra with the ratio of 0.195:1. (d) Merging image of upper and lower inset. Upper inset (blue) and lower inset (red) are flow cytometer dots plot of type-B beads with two different ratios. (e) The HFI system decoded spectra with ratio of 0.237:1. (f) The HFI system decoded spectra with ratio of 0.315:1.

application, the experiment that immobilizes the ssDNA-QDs (Wuhan Jiayuan Quantum Dot Technological Development Corporation) onto the encoded beads and decodes the signal of QD-DNA-PDA@encoded-PS has been done. Figure 10a is the schematic illustration of the process of ssDNA immobilization and signal decoding. First, a layer of polydopamine was coated on the surface of the encoded multicolor beads. Next, DNAQDs were immobilized onto the PDA@encoded-PSs. Last, the signal of the DNA immobilized multicolor beads were decoded using the HFI system. Polydopamine Coating of the Encoded Beads. Dopamine in an alkaline solution triggers oxidative polymerization and can form the polydopamine coating on virtually any surface to which proteins, peptides, oligonucleotides, metal ions, or synthetic polymers are able to be attached72−75 due to the abundant active catechol and amine groups. Thus, the modification of encoded beads with the polydopamine (PDA) is an effective approach for the immobilization of amino or thiol functionalized DNA. The surface morphology of the encoded bead after the PDA modification is characterized by the SEM, as shown in Figure 10b, and the inset is the surface morphology of the original PS for contrast. DNA Immobilization. The water-soluble QDs (with the emission wavelength of 526 nm) were modified with carboxyl groups on their surface. The ssDNA sequence is 5′- NH2AGCGATGCACAGAAAATGCTAGTGCTTATGCAGCAAATGC-SH-3′ where the 5′ amino-group is used for the attachment of water-soluble QDs and the 3′ end of the sequence was immobilized onto the surface of polydopaminecoated beads. The DNA-QDs immobilized encoded beads were characterized by a laser confocal microscope (FV1000, Olympus), as shown in Figure 10c. The green ring is the fluorescence emitted by the water-soluble QDs connected to the ssDNA and the red color inside is the fluorescence emitted by the encoding elements (QD585 nm and QD619 nm). The detection result is shown in Figure 10d, the green solid line

Figure 9. Type-A and type-B beads were denoted with solid and dashed lines, respectively. The ratio of the peak of two colors of QDs was (a) 0.55, (b) 0.585, (c) 0.65, and (d) 0.75.

type-B beads in each batch cannot be seen as the different code under the flow cytometry system for the reason that the Color1 and Color2 are unseparable for the flow cytometer. But the encoded type-A and type-B beads in each batch are distinguishable under the HFI system. So more colors of QDs can be used for the HFI system than the flow cytometer, and more channels in spectral dimension can be used under HFI system. According to Nie,25 1000000 nucleic acid or protein sequences can be encoded, if 10 intensity levels and 6 colors of QDs are used. Theoretically, the HFI system allow the use of much more than 6 colors of QDs to encode analytes, which has profound meaning in biological and medical application. QD-DNA-PDA@encoded-PS Detection and Decoding. To demonstrate that the HFI method can be used for practical F


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Figure 10. (a) Schematic illustration of the process of this real sample analysis. (b) SEM image of surface morphology of the encoded bead after the PDA deposition. The inset shows the original PS. (c) Laser confocal microscope image of QD-DNA-PDA@encoded-PS. (d) The decoding result.

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denotes the DNA fluorescent tag. The mauve solid line denotes the emission spectrum of the encoded multicolor beads, and the blue and red line denotes the decoding result of the two color beads. Thus, we demonstrated that the PDA coating can facilitate the DNA immobilization and the method can be used for practical application to decode the signal of the analytes immobilized multicolor beads.

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CONCLUSION In summary, we designed a line scan hyperspectral fluorescence imaging (HFI) system which can not only obtain and decode the fluorescence spectra, but also obtain the spatial information of encoded multicolor beads. The results of system comparison experiment indicates the HFI system has more usable channels in spectral dimension and is more sensitive for the ratio difference between colors than the flow cytometer, which means more codes can be encoded in HFI system. So, with the combination of multichannel and spatial information acquisition ability, HFI system is a promising multiplexed highthroughput system, which can be applied to various biological and medical applications.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 0755 26036873. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was made possible with the financial support from NSFC China (61275188, 8117137, 61361160416, 61308119), science and technology research program of Shenzhen City (CXZZ20140416160720723, GJHZ20140416153558957). G


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