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Multifunctional Encoded Particles for High-Throughput Biomolecule Analysis

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Science  09 Mar 2007:
Vol. 315, Issue 5817, pp. 1393-1396
DOI: 10.1126/science.1134929

Abstract

High-throughput screening for genetic analysis, combinatorial chemistry, and clinical diagnostics benefits from multiplexing, which allows for the simultaneous assay of several analytes but necessitates an encoding scheme for molecular identification. Current approaches for multiplexed analysis involve complicated or expensive processes for encoding, functionalizing, or decoding active substrates (particles or surfaces) and often yield a very limited number of analyte-specific codes. We present a method based on continuous-flow lithography that combines particle synthesis and encoding and probe incorporation into a single process to generate multifunctional particles bearing over a million unique codes. By using such particles, we demonstrate a multiplexed, single-fluorescence detection of DNA oligomers with encoded particle libraries that can be scanned rapidly in a flow-through microfluidic channel. Furthermore, we demonstrate with high specificity the same multiplexed detection using individual multiprobe particles.

The ability to quantify multiple proteins, cytokines, or nucleic acid sequences in parallel using a single sample allows researchers and clinicians to obtain high-density information with minimal assay time, sample volume, and cost. Such multiplexed analysis is accompanied by several challenges, including molecular encoding and the need to retain assay sensitivity, specificity, and reproducibility with the use of complex mixtures. There are two broad classes of technologies used for multiplexing: planar arrays (13) and suspension (particle-based) arrays (421), both of which have application-specific advantages. Planar arrays, such as DNA and protein microarrays, are best suited for applications requiring ultra-high-density analysis. In comparison, suspension arrays benefit from solution kinetics, ease of assay modification, higher sample throughput, and better quality control by batch synthesis (22). Although particle-based arrays have been used for high-density genotyping applications (23), they are most favorable over microarrays when detecting a modest number of targets over large populations or when rapid probe-set modification is desired. Whereas planar arrays rely strictly on positional encoding, suspension arrays have used a great number of encoding schemes that can be classified as spectrometric (411), graphical (1216), electronic (1719), or physical (20, 21).

Spectrometric encoding uses specific wavelengths of light or radiation [including fluorophores (47), chromophores (8), photonic structures (9), and Raman tags (10, 11)] to identify a species. Fluorescence-encoded microbeads (47) can be rapidly processed by using conventional flow cytometry [or on fiber-optic arrays (24)], making them a popular platform for multiplexing. However, there are several disadvantages of using multiple fluorescent signals as means of barcoding, including (i) the limited barcodes achievable (typically ∼100) because of spectral overlap, (ii) the lack of portability for bulky flow cytometers, (iii) added cost with each fluorescent exciter and detector needed, and (iv) potential interference of encoding fluorescence with analyte-detection fluorescence. For these reasons, single-fluorescence methods exist that use graphical techniques to spatially embed barcodes on microcarriers.

Graphical barcodes rely on the patterning of optical elements on a microcarrier; some examples include striped rods (12, 13), ridged particles (14), and dot-patterned particles (14, 15). The chemistries used to fabricate such particles (metallic or photoresist) require additional coupling chemistries to conjugate biomolecules to the surface, and, in the case of striped rods, each metallic pattern needs to be generated one batch at a time. Typically, the patterns on these particles can only be distinguished if the fluorescence of the target signal is sufficiently high. Another graphical method for microcarrier encoding is the selective photobleaching of codes into fluorescent beads (16). In this method, both particle synthesis and decoding are time-consuming, making it an unlikely candidate for high-throughput analysis. A method that eliminates fluorescence altogether uses radio frequency memory tags (1719). This approach is very powerful because it allows for nearly unlimited barcodes (>1012) and decouples the barcoding scheme from analyte quantification (fluorescence), but the synthesis of any appreciable number (thousands or millions) of these electronic microchip-based carriers may prove to be expensive and slow. These and several other methods developed for multiplexed analysis have been thoroughly reviewed elsewhere (25, 26).

We introduce a technique that overcomes many of these multiplexing limitations. By exploiting laminar flows characteristic of microfluidics, we demonstrate the ability to generate multifunctional particles with distinct regions for analyte encoding and target capture (Fig. 1). In a typical experiment, we flowed two monomer streams (one loaded with a fluorescent dye and the other with an acrylate-modified probe) adjacently down a microfluidic channel and used a variation of continuous-flow lithography (27) to polymerize particles [with 30-ms bursts of ultraviolet (UV) light] across the streams (28) (movie S1). In this manner, particles with a fluorescent, graphically encoded region and a probe-loaded region can be synthesized in a single step. Each particle is an extruded two-dimensional (2D) shape (Fig. 1B) whose morphology is determined by a photomask that is inserted into the field-stop position of the microscope and whose chemistry is determined by the content of the coflowing monomer streams. The cross-linked polymer particles then flow down the channel [without sticking due to oxygen inhibition near the channel surfaces (27)], where they are collected in a reservoir. The particles can be rinsed of excess monomer and then used for biological assays.

Fig. 1.

(A) Schematic diagramofdot-coded particle synthesis showing polymerization across two adjacent laminar streams to make single-probe, half-fluorescent particles [shown in (B)]. (C) Diagrammatic representation of particle features for encoding and analyte detection. Encoding scheme shown allows the generation of 220 (1,048,576) unique codes. (D) Differential interference contrast (DIC) image of particles generated by using the scheme shown in (A). (E to G) Overlap of fluorescence and DIC images of single-probe (E), multiprobe (F, bottom), and probe-gradient (G, left) encoded particles. Shown also is a schematic representation of multiprobe particles (F, top) and a plot of fluorescent intensity along the center line of a gradient particle (G, right). Scale bars indicate 100 μm in (D), (F), and (G) and 50 μmin(E).

We used poly(ethylene glycol) (PEG) (well known as a bio-inert polymer) as the particle foundation to eliminate the need to “block” surfaces after probe conjugation and as a transparent material to allow transmission of fluorescent signal from both particle faces. These properties should enhance both specificity and sensitivity of analyte detection. We used a simple dot-coding scheme to generate particles that can bear over a million (220) codes (Fig. 1C). Particles were designed to be “read” along five lanes down their length, with alignment indicators that were used to identify the code position and the read “direction” despite the particle orientation (Fig. 1C). The flat, long shape of the particles helps align them for scanning in a flow-through device. The spatial separation of various chemistries on the particles allows decoding and target detection to be achieved by using a single fluorophore.

To demonstrate the versatility of particle synthesis, we selectively labeled monomer streams with a fluorophore and used a variety of channel designs to generate particles bearing a single probe region, multiple probe regions, and probe-region gradients (Fig. 1, E to G). Multiprobe particles (Fig. 1F), made with the use of channels with several inlet streams, allow for a direct, single-particle comparison of several targets. Furthermore, probe gradients (Fig. 1G), made by simply allowing diffusion of the probe across streams in a long channel, are useful for broadening the detection range of an analyte when using a fixed detection sensitivity (when the signal can saturate). If magnetic nanoparticles are incorporated in a gradient, it may be possible to produce a temperature variation along particles when stimulated in an oscillating magnetic field (29).

A key feature of our method is the direct incorporation of probes into the encoded particles. This is accomplished by simply adding acrylate-modified biomolecules into the monomer solution. After polymerization, the probes are covalently coupled to the polymer network. This process is applicable for both oligonucleotide and protein probes (3032). We demonstrate that the short bursts of UV used to synthesize probe-conjugated particles are not detrimental to the functionality of incorporated oligonucleotides. Previously, we showed similar results with bead-bound antibodies that were incorporated into polymer structures made from nearly identical monomer constituents (28, 33).

To demonstrate multiplexing capabilities, we used acrylate-modified oligonucleotide probes (which are commercially available) for DNA sequence detection (Fig. 2, A to C). We synthesized three batches of particles: one of which was loaded with 20–base pair (bp) oligonucleotide probe 1 (5′-ATA GCA GAT CAG CAG CCA GA-3′), another with probe 2 (5′-CAC TAT GCG CAG GTT CTC AT-3′), and a third with no probe, to serve as a control. Targets were fluorescently labeled oligonucleotides with complementary sequences to the two probes. We mixed the particles and incubated them for 10 min at room temperature in microwells containing either target 1 (at 1 μM), target 2 (at 1 μM), both targets (both at 0.5 μM), or no target (28). A positive target detection was indicated by probe-region fluorescence, which was more pronounced near the particle edges. This result suggested that targets were able to diffuse and hybridize several μmintothe particle body (28). In each instance, the particles showed uniformity (28) with high specificity to the oligomers, exhibiting fluorescence only when the target was present (Fig. 2C).

Fig. 2.

Multiplexed analysis using single-(A to C) and multiprobe (D to F) encoded particles. The particles were loaded with DNA oligomer probes (O1, 5′-ATA GCA GAT CAG CAG CCA GA-3′, or O2, 5′-CAC TAT GCG CAG GTT CTC AT-3′) or no probe [negative control (C)] as shown schematically in (A) and (E). Shown are representative fluorescence images for single-probe (B) and multiprobe (D) particles after a 10-min incubation with both fluorescent-labeled targets. Fluorescence in the probe regions indicates target detection. Also shown are individual particles after incubation in solutions containing no targets, target 1 only, target 2 only, or both targets [(C) and (F)]. Scale bars, 100 μm.

To further demonstrate the power of our multiplexing scheme, we performed the same sequence detection assay with the use of particles with multiple adjacent functionalities (Fig. 2, D to F). In this manner, we were able to simultaneously assay for the two target sequences (with a negative control) on a single particle. Again, the assay was highly specific (Fig. 2F) and very uniform from particle to particle (Fig. 2D) (28). The interfaces between probes on the particles are very sharp, and thinner stripes could be used for even greater multiplexing capabilities.

In order to prove that this method of multiplexed analysis is practical for high-throughput applications, we developed a simple scheme to scan particles in a flow-through device (Fig. 3). Multiprobe particles used in the hybridization experiment just described (Fig. 2, D to F) were flowed through a microfluidic channel and observed on an inverted fluorescence microscope (28). Particles were aligned by using flow-focusing and traveled down a channel only slightly larger than the particle width (Fig. 3A). We used a biofriendly surfactant (28) to ensure that the particles flowed smoothly down the channels without sticking. Images were taken at a designated detection region in the channel with an exposure of 1/125 s as the particles passed the field of view (using a 20× objective). Image sequences were later analyzed to determine the particle code and quantify targets (movie S2).

Fig. 3.

Flow-through particle reading. (A) Schematic representation of a flow-focusing microfluidic device used to align and read particles after hybridization experiments. Particles are directed down a narrow channel and are imaged by using fluorescence microscopy. (B) A typical image of a particle taken in aflow-through device as shown in (A). The image was captured by using a microscope-mounted camera with an exposure of 1/125 s as the particle flowed at a velocity of ∼1200 μm/s through the channel. Scans of fluorescent intensity were taken across the five lanes of the particle to reveal the code and detect oligomer targets (O1 and O2). With the particle in this orientation, the code is read from right to left and top to bottom, where 1, 0, and x represent a hole, no hole, and an alignment marker, respectively. Particle shown is 90 μm by 270 μm. A.U., arbitrary units.

A representative particle image is shown (Fig. 3B) with corresponding intensity plots along the five particle “reading lanes.” The code along each lane can be determined by analyzing the sharp dips and plateaus in the intensity plots. By using the control-region fluorescence, we defined a positive target detection as the control average intensity plus three standard deviations for each particle. We were able to accurately identify the presence of both oligonucleotide targets after only a short 10-min incubation.

The throughput of our system is primarily determined by the detection scheme and the particle size. The particles synthesized for this study are relatively large compared with those in other flow-through methods, measuring 90 μm in width, ∼30 μm in thickness, and 180 to 270 μm in length. Large size not only limits the throughput of a system but also increases the sample volume. However, the great particle-to-particle reproducibility we have demonstrated (28) will afford a much lower redundancy than is typical in flow-through systems, improving efficiency. By using conservative estimates, we found that our system should be capable of providing rapid, high-density analysis with a manageable sample volume (28) despite the seemingly large particle size.

In addition to being very reproducible, we have also shown that our system is very sensitive. With 30-min incubations, we can detect DNA oligomers comfortably at 500 attomoles without biotin-avidin–aided signal amplification (28). This leads us to believe that our system will be at least as sensitive as current, commercially available multiplexing technologies, with the added advantages of all-in-one particle synthesis, incorporation of multiple probes, low cost (28), virtually unlimited codes, and implementation using little more than a standard fluorescence microscope.

Supporting Online Material

www.sciencemag.org/cgi/content/full/315/5817/1393/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S3

References

Movies S1 and S2

References and Notes

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