Aqueous Two-phase Separation Research Articles

Dual-Catalysts-Embedded spontaneously propelling asymmetric micromotors using triple emulsion microfluidic synthesis for highly efficient nano/microplastic removal

Plastics, which are pivotal in modern technological progress and have a broad range of applications, now pose a significant environmental and health risk globally. Despite their detrimental environmental effects, there is a lack in research on effective plastic removal and treatment strategies. Addressing this urgent issue necessitates the development of innovative and efficient materials and methods for removing plastics from contaminated environments. In this study, we utilized a microfluidic technique to create novel and eco-friendly asymmetrically structured micromotors made of glucose oxidase (GOx)/catalase (Cat) embedded in polyethylene glycol diacrylate (PEGDA). These micromotors are specifically engineered to efficiently remove substantial amounts of plastics from ecosystems. The microfluidic process enabled the production of micromotors with uniform yet asymmetric shapes through aqueous two-phase separation and UV polymerization. By incorporating functional nanomaterials, these micromotors can be customized for specific wastewater treatment needs. Our design utilizes GOx and Cat to consume glucose as fuel, generating oxygen microbubbles. These bubbles not only propel the micromotors but also capture suspended plastic nano/micro-particles, effectively removing them from aquatic environments. The high surface tension of the bubbles allows for the entrapment of plastics at their interface, facilitating their removal from the water. In addition, non-toxic nature of these GOx/Cat PEGDA micromotors makes them especially suitable for environmental remediation and biological applications, addressing the urgent need for environmentally benign solutions to plastic pollution.

Fabrication of Crescent Shaped Microparticles for Particle Templated Droplet Formation.

Crescent-shaped hydrogel microparticles are shown to template uniform volume aqueous droplets upon simple mixing with aqueous and oil media for various bioassays. This emerging "lab on a particle" technique requires hydrogel particles with tunable material properties and dimensions. The crescent shape of the particles is attained by aqueous two-phase separation of polymers followed by photopolymerization of the curable precursor. In this work, the phase separation of poly(ethylene glycol) diacrylate (PEGDA, Mw 700) and dextran (Mw 40000) for tunable manufacturing of crescent-shaped particles is investigated. The particles' morphology is precisely tuned by following a phase diagram, varying the UV intensity, and adjusting the flow rates of various streams. The fabricated particles with variable dimensionsencapsulate uniform aqueous droplets upon mixing with an oil phase. The particles are fluorescently labeled with red and blue emitting dyes at variable concentrations to produce six color-coded particles. The blue fluorescent dye shows a moderate response to the pH change. The fluorescently labeled particles are able to tolerate an extremely acidic solution (pH 1) but disintegrate within an extremely basic solution (pH 14). The particle-templated droplets are able to effectively retain the disintegrating particle and the fluorescent signal at pH 14.

Spontaneous compartmentalization in chitosan - phytate - lignosulfonate microparticles by gas-shearing microfluidics

Compartmentalized microparticles fit a niche in many applications due to their hierarchical structures and designable multifunction. However, it is difficult to avoid using surfactants or other auxiliary components in existing aqueous two-phase or other phase separation systems, which limit the potential bio-applications. Herein, spontaneous compartmentalized chitosan (CS) microparticles with various structures were designed for the first time through gas-shearing microfluidics. It was formed based on the difference in the degree of cross-linking between CS and various cross-linkers. In our design, compartmentalized microparticles only contain CS, phytate (PA) and lignosulfonate (LS), and no other additives are used in the fabrication process. The concentrations of PA and LS affected the structure of microparticles, and crosslinking time and incubation time also had a hold on the compartmentalized behavior of CS-PA-LS microparticles. CS microparticles created using other ionic crosslinkers can also spontaneously compartmentalize based on this strategy. Furthermore, the pH-responsive, antibacterial, and biocompatible properties make CS-PA-LS microparticles attractive materials for widespread applications.

DNA-Surfactant Exchange and Quantifying Interactions of Pure (n,m) Single-Enantiomer SWCNTs Towards Biohybrid Self-Assembly

Self-assembled biohybrid nanostructures made from single-wall carbon nanotubes (SWCNTs) and DNA are a promising model system for studying fundamental photo physics phenomena, and have significant potential as building blocks for applications such as biosensing and photovoltaics devices. Precise control of SWCNT bandgap and length in these biohybrid assemblies is highly desirable, but scalable production of such samples often benefits from processing in aqueous polymer and surfactant solutions that are then typically incompatible with DNA-directed assembly. DNA exchange is a promising route for replacing surfactants with self-assembling DNA sequences after these purification steps. Unfortunately, despite advances, DNA exchange remains a challenging technique with limitations due to uncontrolled aggregation and low yields. In this work we focus on improving the process for several well-defined SWCNT samples that are both single chirality and single enantiomer refined through aqueous two-phase separation as well as length sorted to a narrow distribution. With these, we demonstrate improvements in DNA exchange in both yield and control of the aggregation state by modifying DNA exchange processing parameters. We also show that the exchanged DNA-wrapped SWCNTs can be linked together by the addition of a complementary DNA bridge to form self-assembled biohybrid nanostructures for energy transfer studies. We use analytical ultracentrifugation and absorbance and photoluminescence spectroscopies to measure and quantify changes in as these biohybrid structures self-assemble. These developments pave the way for high yield assembly of precise biohybrid nanostructures for energy transfer studies and open potential applications in photovoltaics and biosensing.

(Invited) Demonstration of ATPS Phase Separation, B-Field Concentration and Manipulation of Sars-Cov-2 Viral Particles

A technology, based on fluorescence sensing in a liquid sample and capable for an early detection of a pathogen at extremely low concentration, is very desirable though equally challenging to develop. In optical sensors, a typical fluorescent signal would be too weak to measure without special tools, unless the detected analyte, tagged with a fluorescent label, can be separated and concentrated.In this work a new combined technology[1] will be presented. An assembly of a monoclonal antibody (mAb) functionalized with a fluorescent tag with the S-proteins at the surface of inactivated coronavirus is achieved. In order to separate a bound fluorescent tag (signal) and free tag molecules (background), we simultaneously attach to the virus surface ferrite magnetic nanoparticles (mNP) functionalized with the mAb (without fluorescent tag). Since the surface of the SARS-CoV2 virus has a large number of binding sites, a "sandwich" assembly of mNP-mAb:virus:mAb-FL happens. At the same time, non-specific binding of the fluorescent tag to mNPs is excluded. As a result, the fluorescent tag bound to the analyte can be manipulated by magnetic force and moved with respect to (unused) free tag molecules.Fig.1 shows the fluorescence signal of the mNP-mAb:virus:mAb-FL sandwich assemblies, that were first concentrated by an aqueous two-phase system separation (ATPS)[2] – carefully designed mix of PEG/dextran solutions. Upon dividing the ATPS into PEG bulk phase and dextran bubbles, the viral assemblies (and free non-bound tag) were separated into the PEG phase. Applying vertical magnetic field as shown in the figure resulted in additional significant concentration of the assemblies against the wall of a dextran bubble. Thus, we achieved a clear separation of the virus (as detected by direct fluorescence signal) with respect to the free tag molecules (background) residing in the bulk solution.[1] Vyacheslav V. Rotkin, Ming Zheng, Tetyana Ignatova, Daniel Hayes, Suresh Kuchipudi; Methods and systems for early detection of viral diseases. https://patents.google.com/patent/US20210364517A1[2] Albertsson, P. A., Partition of Cell Particles and Macromolecules. first ed.; Wiley: New York, 1986. Acknowledgement: This work has been supported by NSF (CHE-2032582 and CHE-2032601). Figure 1

Nanosilicate-hydrogel microspheres formed by aqueous two-phase separation for sustained release of small molecules

Introduction: Hydrogel microspheres are an attractive option for drug delivery applications due to their ease of injection and potential for tunable controlled delivery. However, their utility is limited due to high initial burst release and rapid overall release, which is especially pronounced for small molecules or small size microspheres. We and others have shown that the addition of two-dimensional nanosilicate (NS) particles to hydrogels can significantly prolong release kinetics from hydrogels while minimizing burst release.Materials and Methods: Here we explored whether NS could modulate release kinetics of small molecules from small size injectable microspheres. Polyethylene glycol (PEG)-based hydrogel microspheres were fabricated via polymer/salt aqueous two-phase separation (ATPS), which is facile, high yield, and scalable, without the need for organic solvents or oils.Results and Discussion: Importantly, NS and acridine orange (AO), a model cationic small molecule, were shown to phase separate into the PEG-rich phase, allowing for successful encapsulation within hydrogel microspheres. The fabricated microspheres were stable, similar in size to red blood cells, and easily injectable. The effect of various fabrication parameters, including the addition of NS and AO, on microsphere size and polydispersity were explored. Release of AO was significantly slowed from PEG-NS microspheres compared to PEG-only microspheres and correlated with NS concentration. Two additional small molecules, the chemotherapeutic doxorubicin (positive charge), and the model small molecule Brilliant Blue FCF (negative charge), were shown to exhibit prolonged release, underscoring the broad utility of the system. The dependence of release kinetics on encapsulated NS concentration allows for tunable and prolonged release of small molecules from an injectable hydrogel delivery device.

Abstract 1709: Sustained Release of Small Molecules from Nanocomposite Hydrogel Microspheres formed by Aqueous Two-Phase Separation

Abstract 1709: Sustained Release of Small Molecules from Nanocomposite Hydrogel Microspheres formed by Aqueous Two-Phase Separation

High-Yield Separation of Extracellular Vesicles Using Programmable Zwitterionic Coacervates.

Programmable coacervates based onzwitterionic polymers are designed as dynamic materials for ion exchange bioseparation. These coacervates are proposed as promising materials for the purification of soft nanoparticles such as liposomes and extracellular vesicles (EVs). It is shown that the stimulus-responsiveness of the coacervates and the recruitment of desired molecules can be independently programmed by polymer design. Moreover, the polymeric coacervates can recruit and release intact liposomes, human EVs, and nanoalgosomes in high yields and separate vesicles from different types of impurities, including proteins and nucleic acids. This approach combines the speed and simplicity of precipitation methods and the programmability of chromatography with the gentleness of aqueous two-phase separation, thereby guaranteeing product stability. This material represents a promising alternative for providing a low-shear, gentle, and selective purification method for EVs.

Authenticity and traceability of goat milk: Molecular mechanism of β-carotene biotransformation and accessibility

The efficiently extraction and accurately quantify of β-carotene and its metabolites are crucial for authenticity and traceability in goat milk. Nevertheless, its reliability can be largely improved. In this study, meticulously designed native ESI-MS, fluorescence spectroscopy and molecular docking in combination with cold-induced acetonitrile aqueous two-phase separation system weaken the interaction between β-lactoglobulin and β-carotene metabolites and realized the efficiently extraction. Furthermore, established non-targeted quantitative metabolomics with optimal ion source and variable data-independent acquisition minimized the matrix effects and potential ion suppression. Validated atmospheric pressure chemical ionization-ultra high performance liquid chromatography-Orbitrap method showed that β-carotene as distinctive biomarker in cow milk, and retinol, retinaldehyde, retinoic acid and abscisic acid in goat milk. Collectively, the proposed method is a powerful tool to detect cow adulteration risks in goat milk samples and provides valuable information for availability on authenticity of goat milk.

The role of the bile salt surfactant sodium deoxycholate in aqueous two-phase separation of single-wall carbon nanotubes revealed by systematic parameter variations

Aqueous two-phase (ATP) extraction has been demonstrated as a fast, scalable, and effective separation technique to sort single-wall carbon nanotubes (SWCNTs) according to their diameter and chiral structure. The exact mechanism behind the chirality-dependent migration of SWCNTs between the two phases is however not completely understood, and depends on many parameters (e.g., choice of surfactants and their concentration, pH, temperature, …), making it difficult to optimize the multivariable parameter space. In this work, we present a systematic study of the choice and concentration of specific surfactants on the ATP sorting, by performing a series of single-step ATP separations in which each time only one parameter is systematically varied, while monitoring the structure-specific migration of every SWCNT chirality between both phases with detailed wavelength-dependent spectroscopy. These systematic studies reveal that the diameter-dependent stacking of a discrete number of sodium deoxycholate molecules fitting around the SWCNT circumference determines the separation order in the form of a periodically modulated pattern as a function of SWCNT diameter. Addition of cosurfactants can be used to compete with the bile salt surfactant to enhance the separation yields, but does not affect the sorting order. The results are afterwards directly applied to predict the parameters required to separate specific chiral structures in just two ATP steps.

Aqueous Two-Phase Separation (ATPS) Methods for Oleic acid extraction from Neem leaves

The aqueous two-phase separation system (ATPS) signifies an environmentally responsible approach for the extraction of bioactive compounds from a plants basis, as it is a liquid-liquid fractionation technique centred on the inconsistency of two aqueous solutions. In this investigation, various experimental parameters are optimized as the speed of agitation (200, 300 400 and 500 rpm) and solvent ratio (1:1, 2:3 and 3:2) with 20 % (w/w) of Ammonium Sulphate (AMS) salt composition and 30 % (w/w) of Polyethelyene Glycol (PEG). The obtained extract contains alkaloids, flavonoids, tannins, glycosides, acids and total phenolic compounds (TPC). The extracted Oleic acid by the ATPS method was measured with gallic acid equivalent (GAE) of TPC extracted from neem leaves powder. The determined concentration of oleic acid in the practice of TPC is 8.033 mg of GAE/g from the optimized experimental parameter. The optimized results can be cast off for a commercial process on an industrialized scale. Also, the mathematical modelling investigation was done to intent the critical impeller speed (Njs) with the Zwittering model. The identified model calculates the essential speed of agitation (rpm) for maximum extraction yield.

Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

Accurate Quantification of Sulfonamide Metabolites in Goat Meat: A New Strategy for Minimizing Interaction between Sheep Serum Albumin and Sulfonamide Metabolites.

To date, the determination of sulfonamide metabolites in animal-derived food has universal disadvantages of low throughput and no integrated metabolites involved. In this study, a powerful and reliable strategy for high-throughput screening of sulfonamide metabolites in goat meat was proposed based on an aqueous two-phase separation procedure (ATPS) combined with ultrahigh-performance liquid chromatography quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap). Noncovalent interactions including van der Waals force, hydrogen bonding, and hydrophobic effect were determined to be staple interactions between the sulfonamide metabolites and sheep serum albumin by fluorescence spectroscopy and molecular docking technology, and an 80% acetonitrile-water solution/(NH4)2SO4 was used as ATPS in order to release combined sulfonamide metabolites and minimize the influence of sheep serum albumin. Sulfonamide metabolites in the matrix were screened based on a mechanism of mass natural loss and core structure followed by identification combined with the pharmacokinetic. The developed strategy was validated according to EU standard 2002/657/EC with CCα ranging from 0.07 to 0.98 μg kg-1, accuracy recovery with 84-107%, and RSDs lower than 8.9%. Eighty seven goat meat samples were used for determination of 26 sulfonamides and 8 potential metabolites. On the basis of the established innovative process, this study has successfully implemented the comprehensive detection of sulfonamide metabolites, including N4-acetylated substitution, N4-hydroxylation, 4-nitroso, azo dimers, oxidized nitro, N4 monoglucose conjugation, β-d-glucuronide, and N-4-aminobenzenesulfonyl metabolites, which were shown to undergo oxidation, hydrogenation, sulfation, glucuronidation, glucosylation, and O-aminomethylation.

UV-responsive micellar systems and aqueous two-phase systems based on cationic ester-containing gemini surfactant and sodium trans-ortho-methoxycinnamate

Three ester-containing gemini surfactants (CmDEGs) were used to construct UV-responsive micellar systems with sodium trans-ortho-methoxycinnamate (trans-NaOMCA) as photo-sensitive additive. The effect of tail-chain length and spacer chain length on the UV-responsive ability was preliminarily explored. Longer tail chain and shorter spacer chain are favorable factors for enhancing UV-responsive ability. The addition of trans-NaOMCA into N,N′-bis(2-(dodecanoyloxy)ethyl)-N,N,N′,N′-tetramethyl-1,3-propane diammonium dibromide (C12DEG3) aqueous solution leads to a peak in zero-shear viscosity (η0) versus composition curve, and its further addition induces aqueous two-phase separation. For the C12DEG3/trans-NaOMCA/H2O mixed micellar systems, the UV responsiveness of significant decrease in η0 ascribes to microstructural transitions arising from the changes in counterion binding modes due to the trans-cis photoisomerization of NaOMCA. In comparison with the gemini surfactant 12-3-12, C12DEG3 has ester groups introduced in its tail chains. The introduction of ester groups leads to a significant effect on enhancing the photo-responsive ability in viscosity decrease of the constructed UV-responsive micellar systems. Furthermore, the effect of the addition of an amino acid–based surfactant sodium lauroylglutamate (SLGlu) into the C12DEG3/trans-NaOMCA/H2O mixed system on the UV-responsive behaviors was explored. In addition to UV-induced significant decrease in η0 and phase transition from aqueous two-phase systems (ATPS) to isotropic single-phase systems, the addition of SLGlu can also cause UV-induced increase in η0 and lead to richer UV-induced phase transition behaviors. The reason leading to these differences due to the presence of SLGlu was discussed based on 1H NMR spectra, molecular dynamics (MD) simulation, and molecular electrostatic potential (MEP) analysis.

Photosynthetic hydrogen production by droplet-based microbial micro-reactors under aerobic conditions

The spontaneous self-assembly of multicellular ensembles into living materials with synergistic structure and function remains a considerable challenge in biotechnology and synthetic biology. Here, we exploit the aqueous two-phase separation of dextran-in-PEG emulsion micro-droplets for the capture, spatial organization and immobilization of algal cells or algal/bacterial cell communities to produce discrete multicellular spheroids capable of both aerobic (oxygen producing) and hypoxic (hydrogen producing) photosynthesis in daylight under air. We show that localized oxygen depletion results in hydrogen production from the core of the algal microscale reactor, and demonstrate that enhanced levels of hydrogen evolution can be achieved synergistically by spontaneously enclosing the photosynthetic cells within a shell of bacterial cells undergoing aerobic respiration. Our results highlight a promising droplet-based environmentally benign approach to dispersible photosynthetic microbial micro-reactors comprising segregated cellular micro-niches with dual functionality, and provide a step towards photobiological hydrogen production under aerobic conditions.

Separation and Purification of Papain Crude Extract from Papaya Latex Using Quaternary Ammonium Ionic Liquids as Adjuvants in PEG-Based Aqueous Two-Phase Systems

In this work, the effect of adding different quaternary ammonium ionic liquids (ILs) on the phase formation behaviour of a PEG400+(NH4)2SO4+H2O aqueous two-phase system (ATPS) and separation and purification of crude papain extract from papaya latex in different ATPSs was studied. Phase diagrams were established for different systems: experimental phase diagram data were fitted by the Merchuk equation, and the tie line (TL), composition and tie line length (TLL) were determined. The papain purification effect of ATPSs with various ILs was systematically scrutinized. The absorption spectrum of papain before and after extraction was characterized by UV-vis absorption spectroscopy. SDS-PAGE electrophoresis was used to verify the purity of papain extracted by the ATPSs. Quaternary ammonium ILs as adjuvants did not extend the biphasic area in the phase diagram, but the partition behaviour of crude papain extract in ATPSs changed, resulting in additional IL and papain interactions that promoted papain purification. The highest papain purity was obtained when tetramethyl ammonium bromide ([N1111]Br) was added to the ATPS, and with 20 wt% (NH4)2SO4 and 20 wt% PEG400 at pH 7.0 and 60 °C, the purification factor of crude papain extract was 2.13 and 13.51 and the papain recovery rate was 64.2% and 96.46% for ATPSs with no IL and 4 wt% [N1111] Br, respectively. The UV spectra of papain before and after extraction were the same. The SDS-PAGE analysis revealed that the ATPS containing 4 wt% [N1111] Br enhanced papain purification relative to the ATPS without IL. The addition of 4 wt% IL greatly improved the purification factor of crude papain extract enriched in the PEG phase, providing a potential method for large-scale industrial production of high-purity papain.

Hydrophobin-Protein A Fusion Protein Produced in Plants Efficiently Purified an Anti-West Nile Virus Monoclonal Antibody from Plant Extracts via Aqueous Two-Phase Separation.

The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification.

Spontaneous membrane-less multi-compartmentalization via aqueous two-phase separation in complex coacervate micro-droplets.

Polyelectrolyte/nucleotide multiphase complex coacervate droplets are produced by internalized aqueous two-phase separation and used for the spatially dependent chemical transfer of sugar molecules, providing a step towards the development of membrane-free "organelles" within coacervate-based protocells.

The application of an aqueous two-phase system combined with ultrasonic cell disruption extraction and HPLC in the simultaneous separation and analysis of solanine and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit

An aqueous two-phase system was used in conjunction with ultrasonic cell disruption to extract and separate solanine (mainly solasonine and solamargine) and Solanum nigrum polysaccharide from Solanum nigrum unripe fruit. The optimized conditions of the present study were determined by a single-factor experiment and a multifactor experiment. The concentration of ethanol was set at 60% and the duration of the ultrasonic cell disruption extraction was 50 min. In the ethanol-K2CO3 aqueous two-phase separation system, the concentration of ethanol was 36%, the concentration of K2CO3 was 0.21 mg·mL−1, and the temperature was 15 °C. The solasonine and solamargine were determined by high-performance liquid chromatography, and the Solanum nigrum polysaccharide was determined by an ultraviolet–visible spectrophotometer in accordance with the phenol–sulfuric acid method. xUnder optimized conditions, the average extraction efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 95.86%, 95.95% and 96.95%, respectively, and the average separation efficiencies of solasonine, solamargine and Solanum nigrum polysaccharide were 2.07 mg·g−1, 2.05 mg·g−1 and 8.15 mg·g−1, respectively.

(Invited) Learning to Predict Single-Wall Carbon Nanotube-Recognition DNA Sequences

In this work, we demonstrate the applicability of machine learning methods to discovery of special DNA sequences that recognize partner single-wall carbon nanotubes (SWCNT). DNA/SWCNT hybrids have enabled many applications because of their special ability to disperse and sort SWCNTs by their chirality and handedness. Much work has been done to discover recognition sequences which recognize specific chiralities of SWCNT, and significant progress has been made in understanding the underlying structure and thermodynamics of these hybrids. Nevertheless, de novo prediction of recognition sequences remains essentially impossible and the success rate for their discovery by search of the vast ssDNA library is very low. Here, we report an effective way of predicting recognition sequences based on machine learning analysis of existing experimental sequence data sets. Multiple input feature construction methods (position-specific, term-frequency, combined or segmented term frequency vector, and motif-based feature) were used and compared. The transformed features were used to train several classifier algorithms (logistic regression, support vector machine and artificial neural network). Trained models were used to predict new sets of recognition sequences, and consensus among a number of models was used successfully to counteract the limited size of the data set. Predictions were tested using aqueous two-phase separation. New data thus acquired was used to retrain the models by adding an experimentally tested new set of predicted sequences to the original set. The frequency of finding correct recognition sequences by the trained model increased to >50% from the ~10% success rate in the original training data set.