1.

Introduction

In the recent decade, with the development of many subjects such as life science, chemistry, and so on, a number of compounds and biology molecules have come forth for screening or identifying. How to screen out the target compounds or molecules quickly and with high efficiency from the complex system, which contains a number of candidates, is one of the big problems for the biologist and chemistry analyst.^{1, 2, 3} The analysis technologies, such as electrophoresis and lab-on-chips, make big contributions to the development in the abovementioned fields,^{4, 5} but they are complex to manipulate or have high costs or long time periods and have met the challenges of miniaturization, speediness, and veracity.^{6, 7}

The multiplex and high-throughput analysis based on the decoded beads is a new analysis strategy and has been developed to solve these challenges.^{8, 9, 10} This strategy’s basic principle is that the beads are predecoded by chemical,¹¹ spectral,^{12, 13} electronic, or physical technologies¹⁴ and each coding represents a kind of given information for recognization, at the same time, each of the beads also offers the sites to react with the probes. Before the detection, the different coded beads are linked with different probes such as oligonucleotides or proteins (antibodies), and so each bead has a different probe to capture different targets. The immobilized probes capture the fluorescein-labeled targets in the solution through specific recognizations, and so there appears to be an extra target fluorescent signal in the spectrum of the beads. The beads are excited when passing through the detection channels by microfluids technology, and the signals (including the coding and target signals) of the bead are collected for analysis and decoding, and the results are given.^{12, 15}

In the coding aspect, the optically distinct polystyrene (PS) beads, which are encoded by varying the ratios of red and infrared dyes,^{16, 17} have been reported; each optically encoded bead has a unique spectrum that contains of two emission wavelengths and intensities, which effectually can identify the beads’ encoding type. However, because of the unfavorable properties of the dyes, such as broad emission profiles, spectrum overlapping, and the limited kinds of dyes, the amounts of the beads that can be coded are so far dissatisfactory for the needs. This coding method of 10 intensity levels of two dyes could only yield an array of about 100 beads at the present time.^{18, 19} Compared to the organic dyes, quantum dots (QDs) as coding fluorophores have many unique properties. The intrinsic QDs’ spectral width is about one-third as wide as that of organic fluorescence dyes: the fluorescence intensity of a single $\mathrm{CdSe}$ QD is about 20 times that of a dye molecule.²⁰ Furthermore, the emission wavelength can be tuned continuously by changing the size of the QD particles. Use of these emission-wavelength-different QDs can yield an enormous number of encoded beads, for example, 6 kinds of QDs with 10 intensity levels theoretically can code $1000000$ beads. The most important is that the emission-wavelength-different QD particles can be excited simultaneously by one excitation wavelength due to their the broad-excitation wavelength. That is to say, we can choose the excitation wavelength specific to fluorescent molecules labeling to the target to excite the coding and target signals simultaneously; but for the organic dye–encoded beads, at least two kinds of exciting laser are needed to excite the coding and target signals, respectively.^{16, 17} All of these unique properties are required advantages for optical coding, and therefore, QDs become a perfect luminescent material for optically encoded beads, which have attracted much attention recently.^{21, 22, 23} Recently Nie demonstrated the feasibility of DNA detection using the QD-encoded beads,²² and here we attempted to carry out some trials to put this strategy to practice in a complex system and studied other aspects such as detection range, limitation, sensitivity, and so on.

We even doped the QDs into the porous PS beads by adsorption in solution, thereby encoding the beads, and coated the beads with a thin layer of silica to get precise encodings. In this paper, we immobilized DNA probes to the abovementioned beads and used these beads to detect the fluorescein isothiocynate (FITC)–labeled target DNA sequences in solution and successfully read out the coding and target signals. This work was a successful attempt to study the multiplex and high-throughput analysis and practice based on QD-encoded beads. This technology also provides an alternative approach to detect and analyze specific DNA sequences, and it will be a powerful tool for advanced drug discoveries, disease diagnostics, and biological assays.

2.

Materials and Methods

2.2.

Preparation of Carboxyl Functionalized Beads

Carboxyl functionalized beads $\left(\mathrm{PS}\text{-}\mathrm{COOH}\right)$ were prepared by chlorosulfonation reaction on the surface of beads.²⁴ The 0.5-g PS beads were swollen in $20\mathrm{mL}$ dichloromethane in a flask and stirred for $1\mathrm{h}$ , and then $2\mathrm{mL}$ chlorosulfonic acid was dropped into the flask and stirring continued for $0.5\mathrm{h}$ . Then, only the suspended particles were removed, $10\mathrm{mL}$ 6-amino-caproic acid solution (25% concentration, excessive) were added to the flask, stirred for $24\mathrm{h}$ , and filtered. Then the beads were washed with 1% $\mathrm{HCl}$ , the excess water was removed, and then the beads were dried in a vacuum. This process can be seen in Fig. 1 .

Fig. 1

The sketch of preparation carboxyl functionalized PS beads and covalent of DNA onto the PS beads.

3.

Results and Discussion

The encoding signals of the beads are determined by two main factors: fluorophore emission wavelength and fluorescence intensity if the self-absorption of QDs is negligible. Emission wavelength is the inhesion characteristic of the fluorophore and it has no relation with the quantity of the fluorophores that the bead loaded; but fluorescence intensity is in linear ratio with the quantity of fluorophores that loaded on the bead. Compared with organic dyes, QDs as fluorophore have many unique properties, which are suitable for optical coding. First, because of the broad-excitation wavelength, different emission-wavelength QDs can be excited simultaneously by the same excitation wavelength, that is to say, the excitation wavelength specific to the target-labeled fluorescent molecules could be chosen to excite the coding and target signals simultaneously. In this paper, FITC-labeled target sequences can be seen as study objects, and the FITC excitation wavelength is $480\mathrm{nm}$ , which can also excite the QDs. So, in the following experiments, $480\mathrm{nm}$ was chosen as excitation wavelength to detect the coding $({\lambda }_{QD1em}=576\mathrm{nm}$ , ${\lambda }_{QD2em}=628\mathrm{nm})$ and target $({\lambda }_{FITCem}=520\mathrm{nm})$ signals at the same time. Because the full width at half-maximum of QDs is very narrow (usually about 20 to $35\mathrm{nm}$ ), more coding signals can be contained in the detecting range; this is the second advantage. Third, the QDs have high intensity and stability of fluorescence; the fluorescence intensity of a single $\mathrm{CdSe}$ QD is about 20 times that of the dye molecule.²⁰ Several types of beads were coded in this paper by two different emission wavelength QDs, and the coding spectrums of these beads can be seen in Fig. 2 , for the PS bead $a$ , the normalized intensity ratio (intensity of $576\mathrm{nm}∕\mathrm{intensity}$ of $628\mathrm{nm}$ , the same in the following) is 1:3, for $b$ is 1:1, and for $c$ is 3:1. At the same time, FITC-labeled DNA sequences were chosen as detection targets and the detection principle is showed in Fig. 3 .

Fig. 2

Three spectra of QD-encoded beads. The coding QDs’ emission wavelengths are 576 and $628\mathrm{nm}$ , respectively. The encoding signals are composed of wavelength and intensity. For the PS bead $a$ , the normalized intensity ratio (intensity of $576\mathrm{nm}$ /intensity of $628\mathrm{nm}$ ) is 1:3, for $b$ is 1:1, and for $c$ is 3:1.

Fig. 3

Bead-based array analysis strategy. The beads were encoded by two-color QDs and covalently linked with DNA probes for hybridization array.

As a carrier of bioassays, PS beads have various applications.¹ Considering the encoding reproducibility and feasibility to establish the strategy of detecting target DNA in a complex system, $100\text{-}\mathrm{\mu }\mathrm{m}$ -sized beads were chosed to use as platforms. At $100\mathrm{\mu }\mathrm{m}$ in diameter, the beads present a much larger surface area and higher surface reaction activity than the flat surface.^{29, 30} In order to decrease the steric hindrance and increase the space freedom for DNA probes in hybridization, a “spacer arm” (chlorosulfonic acid and 6-amino-caproic acid) was introduced between the bead and DNA probe, and furthermore, carboxyl functionalized beads can be easily covalently conjugated with biomolecules or probes, which much interest is focused on.^{31, 32} In our former work, the sulfonation group was even introduced to the surfaces of the PS beads by sulfonation reaction, and then grafted with 6-amino-caproic acid as the spacer arm to get $\text{-}\mathrm{COOH}$ functionalized PS beads $\left(\mathrm{PS}\text{-}\mathrm{COOH}\right)$ ; further acid-alkali titration³² results indicated a $\text{-}\mathrm{COOH}$ density of $3.88\mathrm{mmol}∕\mathrm{g}$ .

At the terminus of the spacer arm, the $\text{-}\mathrm{COOH}$ has more space freedom to chemical reactions, and it can be considered that no influence is caused by the beads.³³ Generally, the immobilization of DNA probes is accomplished by abbreviating the probe’s $\text{-}{\mathrm{NH}}_2$ and the $\mathrm{PS}\text{-}\mathrm{COOH}$ bead’s $\text{-}\mathrm{COOH}$ to form stable amide linkage in the existing of EDC and sulfo-NHS.³⁴ In this paper, this method was used to covalently immobilize the DNA probes to the carboxylic beads to detect the target sequences in solution, and the steps can be seen in Fig. 1. Comparing the spectra of the beads before [Fig. 4a ] and after [Fig. 4b] the immobilization of FITC-labeled oligo-DNA0, a clear FITC signal appeared at $520\mathrm{nm}$ . The FITC signal indicates the presence of probes and also proves that the $\text{-}{\mathrm{NH}}_2$ modified DNA can be well covalently immobilized to the carboxyl beads. There are two intentions to introduce the spacer arm molecular 6-amino-caproic acid. First, conjugation efficiency of probes with the spacer arm grafted beads is higher than that with the beads, which have no spacer arm molecules. It was found that the time of reaction with the $\text{-}\mathrm{COOH}$ only needs $10\min$ , while it needs 40 to $60\min$ when directly reacted with the sulfonation group on the surface of the beads, therefore, the reaction time is sharply shortened. Furthermore, by comparing the probe concentration before and after the immobilization in the solution, it can be concluded that the DNA probe density on the $\text{-}\mathrm{COOH}$ spacer arm beads is $3.1\mathrm{mmol}∕\mathrm{g}$ . Considering the original carboxyl density of $3.88\mathrm{mmol}∕\mathrm{g}$ , the covalent efficiency on the spacer arm beads can reach 79.9%, while it can only reach about 40% when the probes were directly immobilized on the beads’ surfaces, which shows that the efficiency is greatly improved. On the other hand, the spacer arm molecules enabled the immobilized probes more space freedom, which is favorable for the next probe-target hybridization.

Fig. 4

Spectra of the carboxyl beads before (a) and after (b) immobilized with FITC-labeled DNA0. The spectra were examined from the beads with the integral time of $40\mathrm{ms}$ . The excitating wavelength is $480\mathrm{nm}$ . Therefore, 576 and $628\mathrm{nm}$ are the encoding signals, and $520\mathrm{nm}$ is the FITC signal.

To study the possibility for the detection of target DNA sequences based on the QD-encoded PS beads, three encoding types of PS beads that immobilized with different probes, respectively, were used in the experiments, named PS-oligo-DNA1 $\left(a\right)$ , PS-oligo-DNA2 $\left(b\right)$ , PS-oligo-DNA3 $\left(c\right)$ . These three types of beads were put into the same hybridization solution that only contains the FITC-labeled $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ sequences (complements of the oligo-DNA2, the probes on $b$ beads), and then the spectra of the three types of beads were detected. Because the target DNA (see Table 1, $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ ), on both its terminals, has no active groups such as $\text{-}{\mathrm{NH}}_3$ to react with the activated $\mathrm{PS}\text{-}\mathrm{COOH}$ , so it was hypothesized that few target DNA could be immobilized in the process of hybridization. After the hybridization, the beads were thoroughly washed with buffers of SSC to ensure that there was no adsorption and nonspecifically bound target DNA on the beads’ surfaces. The results are shown in Fig. 5 . It can be seen that the FITC fluorescence signal did not appear at $520\mathrm{nm}$ in the spectra of beads $a$ and $c$ , while a strong FITC fluorescence signal appeared in the spectra of $b$ . The FITC signal indicates the hybridization of probes with target sequences on the $b$ beads. These results also indicate there was no reaction between the noncomplementary probes and target sequences, so the FITC signal can be detected only on $b$ beads. By varying the target DNA concentration, it could be found that in the range of $0.2\mathrm{\mu }\mathrm{g}∕\mathrm{mL}$ to $30\mathrm{\mu }\mathrm{g}∕\mathrm{mL}$ , the FITC signal intensity is in linear relation with the corresponding target concentration under the same conditions (relative standard deviation $<2\%$ ), as shown in the inset of Fig. 6 .

Fig. 5

Spectra of the beads $(a,b,c)$ that hybridized in the solution containing FITC-labeled oligo- $\mathrm{DNA}{2}^{\prime }$ . Oligo- $\mathrm{DNA}{2}^{\prime }$ is the complement sequence of oligo-DNA2, which immobilized as probe on the bead $b^{\prime }$ ; the sequences are shown in Table 1. The experiment parameters were the same as in Fig. 4.

Fig. 6

Spectra of the bead $b$ hybridized with FITC-labeled oligo- $\mathrm{DNA}{2}^{\prime }$ in different concentrations. Inset shows the relation of FITC signal intensity to the concentration of target oligo- $\mathrm{DNA}{2}^{\prime }$ . The experiment parameters were the same as in Fig. 4.

When the hybridization solution contains $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ and oligo-DNA4, which has the same sequence length but different base pairs, it could be found the beads of type $b$ still captured the FITC-labeled target DNA $\left(\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }\right)$ successfully, which was proved by the obvious FITC signal in the spectrum of curve $b$ in Fig. 7 . Compared with the spectrum of curve $b$ in Fig. 5, the hybridization in the solution that only contains target sequences, the FITC fluorescence signal intensity decreased about 11%. This indicates that the complementary hybridization efficiency decreased. The $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ and the oligo-DNA4 have the same 10-base-pair sequence $\left(5^{\prime }\text{-}\mathrm{CGA}\mathrm{TCG}\mathrm{GGG}\mathrm{CGG}\mathrm{GGC}\right)$ and the oligo-DNA4 possibly partly hybridized with the oligo-DNA2 probe, which disturbed the normal complete hybridization between $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ and oligo-DNA2.

Fig. 7

Spectra of beads hybridized in the solution that contains FITC-labeled oligo- $\mathrm{DNA}{2}^{\prime }$ and oligo-DNA4. Oligo- $\mathrm{DNA}{2}^{\prime }$ and oligo-DNA4 is the same sequence length but the base pairs are different. The experiment parameters were the same as in Fig. 4.

To validate the feasibility of the target DNA detection in a complex solution based on the QD-encoded PS beads, the hybridization properties of the probe-immobilized beads in the denatured calf thymus solutions were studied. The denatured calf thymus DNA solution contains a large number of ssDNA sequences with different base sequence and length and is a perfect complex solution for target DNA detection. The FITC-labeled $\mathrm{oligo}\text{-}\mathrm{DNA}{2}^{\prime }$ in the denatured calf thymus DNA solution, complement to the conjugated probes, can be seen as target DNA; the results are shown in Fig. 8 . The FITC signal appeared only in the spectrum of $b$ that immobilized complementary probes and could be well identified, while the target signal of other types of beads was not detected. However, comparing it with curve of $b$ Fig. 5, hybridization in a single-component solution, the FITC signal intensity decreased 30%, indicating the further hybridization efficiency decreasing of the complementary sequences. This may have been caused by the partial base-pairs hybridization between the probe and short ssDNA that occupied a few bases on the probe sequence and thereby blocked the complete hybridization between target DNA and probe. The partial hybridization of probe and short ssDNA is not stable and dissociation takes place. This case may be overcome by changing certain conditions, such as prolonging the reaction time and washing with the high concentration of salts solution. From Fig. 8, it can be also concluded that, although the target DNA signal can be disturbed by the noncomplementary sequences, this disturbance is not enough to affect the detection. Series experiments validate that the DNA targets can be detected effectively with a detection limit of $0.2\mathrm{\mu }\mathrm{g}∕\mathrm{mL}$ in a complex solution.

Fig. 8

Spectra of the beads that hybridized in the solution contains denatured calf thymus DNA and FITC-labeled oligo- $\mathrm{DNA}{2}^{\prime }$ . The experiment parameters were the same as in Fig. 4.

4.

Conclusions

In this paper, a multiplex analysis technology based on the novel fluorophores QD-encoded beads was systematically studied, and the principles of QD encoding and DNA hybridization analysis were also expatiated. Due to the good optical encoding performances, carboxyl functionalized beads were precisely encoded with two different emission-wavelength QDs in various ratios. Then the differently encoded beads were covalently immobilized with different probes. The target DNA sequences can be successfully detected by using the probe-linked encoded beads in a complex solution. This analysis technology based on QD-encoded beads has advantages in the aspects of sensitivity and portability, and by further improving the detection performances, it will provide a powerful tool in the fields of bioassays, drug discoveries, disease diagnoses, and so on.