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Parallume Encoded Beads

Parallume encoded bead sets, which are available with thousands of optical codes for the multiplexing of any assay, are provided with carboxylate (–CO2H) or amino (-NH2) surface functional groups and in magnetic or non-magnetic formulations.

Parallume | real multiplexing
  1. Introduction to Optically Encoded Beads
    1. Limitations of multiplexed bead technologies
  2. Parallume Encoded Beads
    1. Advantages of Parallume Encoded Beads
  3. Physical and Chemical Properties of Parallume Encoded Beads
    1. General properties of the Parallume Beads
    2. Sensitivity and Dose-Response Data
    3. Bead thermal stability
    4. Bead Sizes and Distributions
    5. Number of Beads Required for Parallume Code and Reporter Resolution
    6. Functional Group Density of Beads
    7. Bead Diffusion Data
  4. Designing the Parallume Multiplex Assay
Parallume Encoded Beads
Fig. 1 A photomicrograph of Parallume-encoded beads using 320 nm excitation. In optical multiplexing, a given probe is attached to a bead with a unique optical signature allowing reactions of that probe to be optically distinguished from other beads possessing other probes when the beads are pooled.

1. Introduction to Optically Encoded Beads

On a DNA or protein microarray the identity of a given probe is determined from its placement during fabrication onto a known x, y location on the planar substrate. To overcome limitations of 2-D arrays, such as poor diffusional mixing and slow reaction kinetics, small sample size (monolayer), limited dynamic range, thermal cycling instability in binding the biomolecular content to the glass substrates and the difficulty in conveniently scaling flat arrays to arbitrarily large number of samples, 3-D bead-based suspension arrays would be very useful and the advantages and benefits of multiplexing in this manner are described in the Parallume Introduction section. To perform multiplexed experiments or assays with optically encoded beads (Fig. 1), a given DNA capture probe, antigen or antibody is attached to a bead with a unique code, the encoded beads with their attached probes pooled and the pooled probe beads screened against a multicomponent sample mixture to determine the presence and amounts of the target species present. As shown in Fig. 2, once the multiplex assay has been performed, the Parallume code of each bead is first determined then the reporter signal level is measured to determine the target concentration.

The Parallume encoding materials are also available in nanoparticle formulations, which have surfaces ready to attach nucleic acid, proteins or small molecules that are suitable for multiplexed histology or pathology studies (see Parallume Nanoparticles).

Parallume code - Capture probe (left) - Reporter signal (right)
Fig. 2 In order to determine the target level in the multiplexed assay, an image of a known Parallume code with a known capture probe attached is obtained (left). The target level in the sample is then determined by measuring the reporter signal intensity in a second image (right).

Current Multiplexing Limitations  Contemporary suspension array multiplexing technology based on optical encoding with organic dyes, which provides <100 resolvable optical codes (see Parallume Introduction), is often performed with dyed polystyrene spheres whose codes and assays are measured with a specialized flow cytometer system. The organic dye-based polymer beads have a variety of shortcomings:

  • Limited number of optical codes (i.e., low multiplexing depth) due to spectral overlap of individual organic dyes
  • Rapid photobleaching of constituent organic dyes
  • Separate excitation source required for each dye
  • Thermally unstable beads cannot be cycled under PCR conditions
  • Low surface area leading to decreased sensitivity and small dynamic range
  • Expensive, specialized instrumentation required for analysis
  • Cannot rerun or archive samples
  • Inefficient bead use; low percentage of total measured beads used for analysis
  • Unnatural, hydrophobic polystyrene surfaces lead to high non-specific binding.

Our Parallume optical encoding technology and materials address all of these shortcomings.

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2. Parallume Encoded Beads

Parallume Optical Bins
Fig. 3 An example of the optical bins created from a 3-color Parallume system. In an experiment, the intensity of the reporter signal for each code gives the amount of target bound to that probe. Note that the majority of the tricolor ratiometric space is still available for the creation of additional optical codes.

The rare earth-encoded Parallume beads (Parallume Introduction) may be used in any type of multiplexed assay or high throughput screening application. A discussion of the types bead’s surface chemical functional groups, a partial list of various applications and protocols to attach proteins, DNA or other molecule to the beads, are found in the Applications and Protocols section. The beads may be (a) incubated, washed, filter, centrifuged, imaged and ultimately archived in Bead Localization Slides as described in Bead Handling Devices, (b) automatically imaged using the Multiplex Assay Reader System (MARS) bead imaging instrumentation and the multiplex data analyzed using the MARSoft software package. We also provide confidential, rapid and cost effective Services to attach your proprietary materials to Parallume encoded bead sets. Additional information concerning the Parallume-encoded beads, bead handling fixtures, imaging systems and software available for purchase may be found in the Purchase section. A discussion of bead imaging statistics may be found in the Literature section.

2.A   Advantages of Parallume Encoded Beads

The advantages and benefits of the Parallume encoded beads for multiplexing, when compared to systems based on polystyrene beads with organic dyes or any other suspension array multiplexing system, are numerous, substantial and diverse. Some of these advantages include:

  • Thousands of Parallume Optical Codes With six or more highly resolvable rare earth encoding colors available, we have statistically demonstrated the resolution of around 10 billion optical codes (Parallume Introduction). Bead sets with certain standard Parallume codes are available (Purchase) as well as custom formulations. Since Parallel manufactures all Parallume-encoded beads in house in Santa Clara CA, any number or type of beads are available. Please Contact us to discuss any special requirements.
  • Ratiometric Parallume Codes Unlike dyed polymer beads, where every bead must be exactly the same size in order to decode the optical codes, the Parallume optical codes are based on ratios and are therefore invariant toward sample size and brightness of illumination source angle of illumination. Furthermore, Parallume only requires a single monochromatic excitation source. The imaged beads are sorted into optical bins for identification based on the ratios of the relative fluorescent intensity from the Parallume emitters (Fig. 3).
  • Parallume Beads Are Synthesized from Agarose Agarose has been used for decades by biochemists and molecular biologists for separations, the synthesis of capture beads and many other applications and its properties have been well documented. The benign, familiar and biochemically inert agarose surface has much lower non-specific binding than the unnatural, hydrophobic polystyrene surfaces of organic dye-based beads. The cross-linked agarose beads are far more stable than polymer beads as discussed below.
  • Extremely Fast Internal Diffusion Rates Similar to the familiar cast agarose gel the Parallume beads are extremely porous and nucleotides up to 800 nt and proteins up to 200-250kDA diffuse throughout most of the pores in the bead. The diffusion rates are very fast resulting in hybridization or binding reactions that reach steady state in minutes not hours (see Fig. 7 below).
  • Magnetic or Non-magnetic Bead Formulations The non-magnetic Parallume-encoded beads are of a size and density that they may be suspended into aqueous solutions by agitation but settle within a minutes from an unstirred solution. For those scientists whose protocols include isolation using magnetic capture beads, the Parallume encoded beads are also available with entrained nanoparticulate superparamagnetic Fe2O3 (see below) which these beads to be washed and manipulated employing standard magnetic bead handling protocols.
  • Parallume magnetic beads
    Parallume encoded magnetic beads shown under visible light (left) and 302nm (right).
  • Familiar Conjugation Chemistries The DNA, proteins or small molecules to be analyzed are usually attached to the Parallume beads via covalent linkages to prevent any attrition or leaching of the biocontent from the beads. There is absolutely no loss of signal when dyed oligos are covalently attached to the bead surface even after 200 PCR cycles. The most popular and generally useful bead surface functionalization moiety is the carboxylate (-CO2H) group and we provide facile and robust protocols for the attachment of content to the beads in the Applications and Protocols section. If you prefer, we provide rapid, confidential and convenient conjugation and bead scanning Services. Third party kits for conjugating biocontent to carboxylated surfaces should be compatible with the Parallume-encoded agarose beads.
  • Absolutely No Photobleaching: Samples May Be Imaged or Stored Indefinitely The solid state inorganic oxides from which the Parallume encoding materials are derived cannot photobleach. Materials of this type are so photochemically stable that they are used to fabricate resonant laser cavities. It is therefore possible to excite and image samples indefinitely, or years later, without any loss whatsoever in fluorescent signal intensity. The Parallume oxides are stable in air up to their melting point of ~1800°C.
  • Single Parallume Excitation Source Required Unlike the organic dye-based multiplexing systems where a separate laser excitation source is required for each encoding dye color, the Parallume materials only require a single monochromatic excitation source. Therefore, changes in optical codes due to excitation power fluctuations, sample to detector distance, misalignment of the laser, angle of illumination or sample size are not possible with the Parallume materials. This results in much more numerous and stable optical codes.
  • Thermally Stable Parallume Beads May Be PCR Cycled The Parallume-encoded agarose beads may be steam sterilized in an autoclave, indefinitely thermally cycled under PCR conditions or frozen in water with absolutely no effect on bead integrity or any evidence of loss or leaching of bead-bound biocontent. Please see the Applications and Protocols section for examples of primer extension, single base extension and clonal amplification.
  • Samples May Be Conveniently Archived The beads are manipulated and imaged in 25mm x 75mm Bead Localization Slides (BLS) as described in the Bead Handling Devices section. These slides can be easily archived using a manual or automated storage and retrieval system for indefinite archival storage.
  • Minimum Bead Handling Losses with 100% Utilization Rates Unlike organic dye-based encoded polystyrene beads, where it is sometime necessary to begin an assay with several thousand beads in order to account for handling attrition and rejected beads, even the smallest number of Parallume-encoded beads may be handled with no losses. This is possible because of the uniquely efficient design of the Bead Localization Slides (BLS) and the BLS fixture as described in the Bead Handling Devices section. Once the beads are placed into the handling apparatus it is possible to incubate, wash, dry, filter, evacuate, centrifuge or image the beads without removing them from the apparatus.
  • High Surface Area and Large Dynamic Range The Parallume-encoded beads, like all agarose beads, provide a high surface area for efficient target capture but the beads are completely transparent to reporter emission thereby providing excellent sensitivity and dynamic range. As a result of the high surface area of the porous agarose beads, very few beads are needed for any assay and good counting statistics may be obtained with as few as 5-20 beads. This contrasts strongly to the organically-dyed polystyrene flow cytometer beads where it is sometimes necessary to screen thousands of beads to obtain enough data to provide acceptable counting statistics.
  • Any Visible or IR Reporters May Be Used There is absolutely no optical cross talk or cross excitation between the Parallume emitters and reporters even if they are the same color. The Parallume beads are excited in the mid-UV (320nm), a wavelength which does not excite either DNA or proteins (absorbing in the 260-280nm range) or colored dyes (absorbing in the visible above 375nm), while, conversely, the visible-light-transparent Parallume phosphors absorb none of the visible excitation wavelengths used to excite the organic reporter dyes. Therefore, since there is no optical crosstalk, any color reporter dyes may be used without regard to color.
  • No Dye Leaching to Alter Optical Codes The Parallume is permanently entrained within the agarose and cannot be removed. The immutable Parallume optical codes cannot be altered once synthesized.

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3. Physical and Chemical Properties of Parallume Beads

This section discusses a variety of chemical properties and physical characteristics of the Parallume encoded agarose beads such as sensitivity for protein-antibody and DNA probe-target assays, thermal stability, size distribution, functional group density, data for target diffusion rates into beads and other data.

Topics discussed include:

3.A   General properties of the Parallume of Beads

After the evaluation of several different potential bead materials including polystyrene, latex, porous glass, agarose and other materials, the beads were fabricated from agarose because of its outstanding chemical and thermal stability, ease of manufacture and control of bead size, a familiar and benign surface and the ability to prepare beads with a high density of functional groups on the bead surface to allow facile biocontent attachment. A summary of select properties of the Parallume encoded agarose beads is included below.

The Parallume encoded beads are synthesized using a proprietary process that incorporates Parallume nanoparticles into the agarose from which the beads are formed. Once formed the beads are chemically derivitized yielding a functional group density = 3-6 µmol/mL of settled beads. The agarose beads are also chemically cross-linked which gives them a thermal stability far beyond that of polystyrene beads and allows the beads to be sterilized by autoclaving, boiled or thermally cycled under PCR conditions or frozen with no observable deleterious effects.

A summary of some physical and chemical properties of the Parallume encoded beads is given in Table 1.

Table 1: Properties of Parallume Encoded Beads

Item or Property
Specification or Value
Parallume encoded bead materials Crosslinked, chemically derivitized agarose encoded with Parallume nanoparticles; magnetic or non-magnetic beads available
Size of beads Size ranges available: <40µ, 40-60µ and >60µ
Number of beads / µL (mL) See Fig. 5 below
Number of functional groups per unit bead volume 3-6 µmol/mL of settled beads
Number of Parallume emitter colors Six available in visible; additional encoding in IR or UV available
Theoretical number of Parallume codes available For 100 binary ratios resolved among six colors 1010 codes available
Thermal stability May be steam autoclaved or PCR cycled
Freezing Beads unharmed by freezing
Compatible reporter colors Any reporter color may be used; no optical cross talk between Parallume and reporters

Parallume Dose-response
Fig. 4 Dose-response data for the titration of a labeled oligonucleotide target onto its bead-bound probe (top) and for detection of IL-6 (bottom).

3.B   Sensitivity and Dose-Response Data

The large surface area and highly porous structure of the Parallume agarose beads provides a large number of potential capture sites (see below) and we have demonstrated that neither the penetration of the UV excitation or emission of the visible light is appreciably attenuated even in beads over 100µ as discussed in the Literature section. The large number of capture sites, combined with fact that the Parallume agarose beads are mostly water and are very porous, means that the beads are both sensitive and display high diffusion rates.

To determine the detection limits of an on-bead DNA hybridization reaction an amine-derivitized oligo was coupled to the carboxylated Parallume beads using standard EDC/NHS and a complementary Cy3-dyed target was stirred with the beads. The dose-response curve for the reaction of a Cy3-labeled target with its complementary bead-bound capture probe is shown in Fig. 4 (top).

To determine the detection limits for a typical on-bead ELISA experiment a monoclonal antibody against IL-6 was covalently bound to a Parallume bead then the beads were exposed to various concentrations of the antigen. Treatment of the antigen-exposed beads with a biotinylated secondary monoclonal antibody against the antigen followed by SAPE staining gives a sensitivity dose-response curve typical of related ELISA detection technologies (Fig. 4 bottom).

3.C   Thermal stability

The cross-linked Parallume encoded agarose are thermally very stable. There is no sign of change in bead morphology after boiling or autoclaving the beads. When a Cy3-dyed amino-derivitized oligonucleotide is coupled to a carboxylic acid group on the Parallume encoded beads using standard EDC/NHS chemistry, and the sample heated and stirred at 98°C, the majority of the bound oligonucleotide is still attached to the beads after one hour as shown in Fig. 5.

3.D   Bead Sizes and Distributions

The Parallume encoded beads are available in various size ranges as described in the Purchase section depending on the application. As explained in the Bead Statistics document found in the Literature section of this website all bead sizes can be used because the Parallume code is derived from the ratio between two emitters which is bead size invariant. However, the reporter
signal in most system requires an absolute, non-ratiometric intensity measurement to give target levels. Since the target intensity would scale with the volume of the bead, all previous optically encoded beads must be manufactured to exacting size standards resulting in a very high cost. In contrast, the Bead Statistics document shows that in the case of Parallume encoded beads, there is a constant weight percentage of Parallume encoding material in each bead so the total integrated Parallume emission intensity can be used to normalize the reporter intensity to a common mean allowing the use of all bead sizes. The bead size and number of beads of various sizes per unit volume (Fig. 6) allows one to calculate the number of moles of functional groups in a given number of beads or a given volume of beads, by calculation, the number of moles of functional groups per unit bead volume.

Parallume Thermal Stability of Oligo on Carboxylated Agarose Beads
Fig. 5 A Cy3-dyed oligonucleotide, which was covalently attached to a Parallume encoded agarose bead, is not removed by heating the sample to 98°C for the indicated times.

Parallume Volume of Bead and Beads per Volume
Fig. 6 The volume of a bead in µm3 (red) and the number of beads/µL (blue) are shown for various bead diameters in microns.

3.E   Number of Beads Required for Parallume Code and Reporter Resolution

Unlike organic dye-encoded polystyrene beads, where thousands of beads may need to be examined in order to obtain a statistically meaningful result, excellent data may be obtained from Parallume encoded beads by using just a few beads. This is due to the combination of a large surface area and high density of capture probes with the high transparency of the beads toward with respect to the emitted light. As shown in Fig. 7, as the number of beads in a BLS well is decreased from >100 beads to three beads, there is no drifting of the optical bins with respect to number of beads measured.

Sorting Parallume Beads into correct Optical Bins
Fig. 7 The ability to sort the Parallume encoded beads into their correct optical bins is independent of the number of beads measured. This data shows that as the number of beads is decreased ultimately down to three beads, that the beads are still resolved into their correct optical bins.

Likewise, the reporter signal (Fig. 8) on the same beads shown in Fig. 7 shows little variance in signal intensity or magnitude of error as a function of the number of beads measured.
It appears from this data that it may be possible under some conditions to obtain meaningful data from a single Parallume encoded bead.

3.F   Functional Group Density of Beads

The most popular and convenient form of functional groups on the surface of the Parallume encoded beads is the carboxylate group. The number of carboxylate groups on the surface of the agarose beads can be obtained by titrating a rapidly stirred slurry of carboxylated Parallume beads with a solution of sodium hydroxide. This titration is shown in Fig. 9 and constitutes a textbook example of a titration of a weak acid with a strong base. Also shown in is a sample where the carboxylate groups have been partially blocked with methoxy-ethanolamine.

3.G   Bead Diffusion Data

The rates at which bead-bound oligonucleotide or antibody capture probes react with their targets is far faster than planar microarrays. As shown in Fig. 10, a concentrated (1 µmol/L) Cy3-dyed 26 nt DNA target diffuses into the pores of a Parallume beads faster than the capture probe laden beads and target solution could be mixed and measured. The resultant hybridization reached steady state in < 2 minutes (i.e., the mixing and measurement time) as depicted in Fig. 10 (top). The steady state concentration of a 100nt target at 1 nM concentration occurs in <20 minutes.

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Fig. 8 There is a relatively small variance in the signal intensity or relative error when the number of beads measured is reduced from >100 to 3.

4. Designing the Parallume Multiplex Assay

Titration of carboxylate Parallume Beads
Fig. 9 Titration of carboxylate Parallume beads shows that there is 3-6µmol of carboxylate groups per mL of beads. Also shown is a sample where a majority of the carboxylate groups have been blocked by coupling with EDC and methoxy-ethanolamine.

It is quick and easy to use the Parallume beads and the MARSoft software to design and build your multiplexed diagnostic protein or nucleic acid assay. The basic outline of the process is given below (Fig. 11). Once you have designed your capture probe – target couples to be used for your particular multiplexed assay (either antigen-antibody or complementary oligo pairs) the time, cost and workflow of the assay is similar to that of a single-plex assay.

In addition to these substantial time and cost savings, there is always an advantage in terms of sensitivity with a multiplex assay as it is not necessary to dilute the target mixture n-fold to perform the n individual single-plex reactions as discussed in the Parallume Introduction. Each probe is attached to an optically encoded Parallume bead set and these probe-conjugated beads pooled to query a multicomponent target mixture.

CY-dyed target stirred with capture probe bound to Parallume Beads
Fig. 10 When a 1µM target solution of a Cy-dyed 26 nt target is stirred with its complementary capture probe bound to Parallume beads the hybridization reaction reached steady state in less than 2 minutes (top), while a 1nM 100nt target requires ~20 minutes (bottom).

More details concerning the types of applications amenable to Parallume multiplexing along with detailed protocols to attach your biocontent and perform the assay are found in the Applications and Protocols sections, additional information about Bead Imaging Statistics is located in the Literature section, the analysis of the bead images for multiplex assays is in the MARSoft section, information regarding the manipulation and handling of the beads is in the Bead Handling Devices area and the types of beads available for purchase found in the Purchase section of this website. Please contact us to discuss our Services for confidential assistance in designing or performing your multiplexed assay or high throughput screening experiment.

As outlined in Fig. 11, the multiplexing of either an antibody-antigen or nucleic acid-based assay follows a similar overall design and execution. In the multiplex assay or experiment a target mixture contains many analytes of interest the amounts of which will all be determined simultaneously within a single reaction vessel. Each DNA sequence or antibody used to detect its corresponding analyte in the target mixture is associated with beads of a unique and resolvable Parallume optical code. Each capture probe, which is either an antibody against an antigen of interest or a nucleic acid sequence complimentary to a DNA/RNA sequence to be detected, is attached to an encoded Parallume bead via standard EDC/NHS chemistry or electrostatically via –NH2+ ammonium groups (Applications and Protocols). The beads, with each capture probe on its own uniquely encoded bead set, are pooled together and the reaction treated as any protein- antibody or hybridization assay.

Multiplex Assay Design and Process Flow for Parallume Beads
Fig. 11 The process flow of designing, building and executing a multiplex antibody or nucleic acid assay using Parallume optically encoded beads.

Examples of some of the reactions possible using the Parallume encoded beads are shown for nucleic acids in Fig. 12 and for protein-antibody reactions in Fig. 13, respectively.

Facile means to multiplex hybridization or any nucleic acid-based assay
Fig. 12 The Parallume Beads (PB) provide a straightforward and facile means to multiplex hybridization or any other nucleic acid-based assay in a variety of ways either in your laboratory or by taking advantage of Parallel’s conjugation Services. The PBs are available with carboxylate (-CO2H) (A) or amino (-NH2) surface functionalities to which capture probes (P) may be covalently attached to the bead (B) via intervening spacer/linkers (L) as shown in (C). The target (T) of the capture probe may be detected on the bead by either using a stained target (D) acting as the reporter or via a tripartite probe-target-reporter ensemble (E). The thermally stable Parallume beads can be thermally cycled or undergo primer extension (F) with no decomposition.

Any antigen-antibody or protein-protein interaction maybe studied in a multiplexed fashion using Parallume encoded beads.
Fig. 13 Any antigen-antibody or protein-protein interaction maybe studied in a multiplexed fashion using Parallume encoded beads. The antibodies or proteins may be attached to the beads via electrostatic or covalent bonding. After the multicomponent antigen target mixture is selected, a different primary capture antibody against each antigen is attached to its own unique Parallume encoded bead set (A), all bead sets pooled together and treated with the target mixture (B). To detect the captured antigen a dye-labeled detection antibody is bound either (a) directly to the antigen (C) or (b) to an antibody which is bound to the antigen (D).

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