Shang-Jung Wu

Conventional techniques for purifying macromolecular conjugates often require complex and costly installments that are inaccessible to most laboratories. In this work, we develop a one-step micropreparative method based on a trilayered polyacrylamide gel electrophoresis (MP-PAGE) setup to purify biological samples, synthetic nanoparticles, as well as biohybrid complexes. We apply this method to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using the conventional crush-and-soak method. MP-PAGE was also able to isolate enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, which is comparable to purities achieved through a more complex two-step purification procedure involving size exclusion and immobilized metal-ion affinity chromatography. This technique was further extended to demonstrate size-dependent separation of a commercial mixture of graphene quantum dots (GQDs) into three different fractions with distinct optical properties. Finally, MP-PAGE was used to isolate DNA-EYFP and DNA-GQD bioconjugates from their reaction mixture of DNA and EYFP and GQD precursors, samples that otherwise could not be effectively purified by conventional chromatography. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

In this study, we develop and apply a directed evolution approach to engineer the optical sensing properties of DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs) towards mycotoxins, a class of molecules critical to detect in the food industry. We successfully demonstrate the creation of sensors for the detection of both the aflatoxin B1 (AFB1) and fumonisin B1 (FB1) mycotoxins based on the specific response of the (9,4) and (7,5) SWCNT chirality fluorescence peaks, respectively. The resulting chirality-specific responsivity was used to demonstrate the multimodal detection of both mycotoxins at different wavelengths of light in the presence of complex food medium. Moreover, we show that directed evolution can be used not only to improve the chiral-dependent selectivity of our sensors to the mycotoxins, but also the sensor sensitivity and fluorescence intensity through multiple rounds of evolution. The approach demonstrated in this study is versatile and could be generalized to other SWCNT sensors as well as other nanosensors comprising a biological element.

Spanning the tissue transparency window, the near-infrared (NIR) photoluminescence (PL) of single-walled carbon nanotubes (SWCNTs) can optically penetrate biological tissue for deep-tissue imaging and optical sensing. SWCNTs are often functionalized with single-stranded DNA (ssDNA) to yield biocompatible, responsive, and selective sensors. However, the low brightness of these ssDNA-wrapped SWCNTs sensors restricts the depth at which such sensors can be implanted in the tissue. This work demonstrates the PL enhancement of ssDNA-wrapped SWCNTs by incorporating biocompatible graphene quantum dots (GQDs). Two kinds of GQDs, pristine (PGQDs) and nitrogen-doped (NGQDs), were fabricated and characterized. Thermodynamically, both GQDs were shown to significantly increase the fluorescence efficiency of ssDNA-SWCNTs with the same degree of PL enhancement after 3 h. Furthermore, a correlation between the diameter of the SWCNTs and the PL enhancement factor was found; the larger the SWCNT diameter, the higher the PL increase upon adding GQDs. For instance, a 30-fold enhancement was achieved for (8,6) chirality while it was only 2-fold for the (6,5) chirality. Our experiments showed that adding GQDs increases the surface coverage of SWCNTs suspended by ssDNA, limiting water molecules’ access to the nanotube surface, thus increasing the fluorescence efficiency. Kinetically, NGQDs brightened SWCNTs much faster than PGQDs. The PL intensity reached a plateau in 2 min following the addition of NGQDs, while it was still increasing even after 1 h upon the addition of PGQDs. We show that NGQDs can act as reducing agents to decrease the amount of dissolved oxygen, which quenches the SWCNTs PL. This advancement provides a promising tool for engineering the brightness of NIR sensors for biomedical applications such as single-molecule imaging of individual SWCNTs using NIR confocal microscopy and deep tissue sensing.

The biochemical and biomedical fields hinge on the ability to effectively separate and purify biological macromolecules. Though this need is largely addressed with a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. In this work, we introduce a simple micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates. Using a conventional vertical slab system, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Finally, we demonstrate the successful isolation of an EYFP-DNA bioconjugate sample that otherwise could not be processed using the two-step chromatography procedure. MP-PAGE thus offers a rapid and versatile means of purifying a variety of biomolecules without the need for specialized equipment.

Semiconducting single-walled carbon nanotubes (SWCNTs) are among the few photostable optical emitters that are ideal for sensing, imaging, drug delivery, and monitoring of protein activity. These applications often require strategies for immobilizing proteins onto the nanotube while preserving the optical properties of the SWCNTs. Site-specific and oriented immobilization strategies, in particular, offer advantages for improving sensor and optical signaling responses. In this study, we demonstrate site-specific protein immobilization of a model enhanced yellow fluorescent protein (EYFP) with a single engineered cysteine residue, using either single-stranded DNA (ssDNA) or a pyrene-containing linker to interact with the SWCNT surface. Protein expression and bioconjugation were characterized using a combination of gel electrophoresis, absorbance, fluorescence, mass spectrometry, and circular dichroism measurements. The results confirm successful protein immobilization to SWCNTs, which retain their near-infrared fluorescence following conjugation. The successful demonstration of these bioconjugation strategies serves as a basis for more cost-effective, site-specific immobilization strategies that can help preserve protein folding and functionality.

The near-infrared emissions of single-walled carbon nanotubes (SWCNTs) falls within the optical transparency window of biological tissue, allowing deep-tissue, in vivo and in vitro imaging. However, the relatively low photoluminescence quantum yield of SWCNTs, which typically lies in the range of 0.1 - 1.5%, limits the penetration depth and sensitivity of these fluorophores when used as imaging agents and optical sensors. In the present work, we enhance the fluorescence emission of SWCNTs up to 10-fold by employing different localized surface plasmon resonance (LSPR) nanostructures. We observe a chirality-dependent enhancement that varies with the nanostructure geometry and material. The proposed mechanism is based on changes in the charge density following nanoparticle conjugation with the SWCNT. In agreement with this mechanism, the enhancement and quenching effects show a strong correlation with the distance between the SWCNTs and the nanostructures. Finally, we demonstrate the applicability of this technology in a single-molecule imaging SWCNT application.

Surfactants offer a tunable approach for modulating the exposed surface area of a nanoparticle. They further present a scalable and cost-effective means for suspending single-walled carbon nanotubes (SWCNTs), which have demonstrated practical use as fluorescence sensors. Though surfactant suspensions show record quantum yields for SWCNTs in aqueous solutions, they lack the selectivity that is vital for optical sensing. We present a new method for controlling the selectivity of optical SWCNT sensors through colloidal templating of the exposed surface area. Colloidal nanotube sensors were obtained using various concentrations of sodium cholate, and their performances were compared to DNA-SWCNT optical sensors. Sensor responses were recorded against a library of bioanalytes, including neurotransmitters, amino acids, and sugars. We report an intensity response towards dopamine and serotonin for all sodium cholate-suspended SWCNT concentrations. We further identify a selective, 14.1 nm and 10.3 nm wavelength red-shifting response to serotonin for SWCNTs suspended in 1.5 and 0.5 mM sodium cholate, respectively. Through controlled, adsorption-based tuning of the nanotube surface, this study demonstrates the applicability of sub-critical colloidal suspensions to achieve selectivities exceed- ing those previously reported for DNA-SWCNT sensors.

DNA has been extensively studied due to its versatility as a dispersant of SWCNTs. The specificity and binding affinity of DNA to various analytes has been shown to be strongly sequence dependent. As a result, through modifications of the nucleotide sequence, the DNA conformation on the SWCNT surface can be tailored to suit specific needs for techniques such as single-molecule detection, in vivo imaging, and chirality separation. To date, the majority of research has focused on the interaction between single-stranded DNA and SWCNT surface, with less focus on the nature of the interaction between double-stranded DNA (dsDNA) and SWCNTs. As a result, the exact interaction mechanism governing this type of complex remains strongly debated and largely unknown. In this study, we employ various biochemical methods to infer the conformation of dsDNA on the surface of SWCNTs. Our methods are based on imaging techniques for identifying DNA-modifying enzyme activity in the presence of dsDNA-SWCNT complexes. Our findings suggest that dsDNA can partially retain its native conformation on the SWCNT surface, and the degree of dsDNA accessibility is strongly sequence dependent. These findings offer new possibilities for SWCNT sensing applications that employ dsDNA, such as the optical detection of DNA-protein interactions.

Directed evolution is a powerful approach for tailoring protein properties toward new or enhanced functions. This technique allows one to alter a protein in a directed manner even in the absence of a known relationship between protein structure and function. Several nanocomplexes, such as optical sensors based on DNA-wrapped single-walled carbon nanotubes (DNA-SWCNTs), similarly lack a known structure-function relationship. For example, DNA-SWCNT complexes exhibit variations in their fluorescence that depend on the sequence of the DNA wrapping. However, little is known about the relationship between the DNA sequence and its effects on the fluorescence properties of SWCNTs. In this study, we use directed evolution as a guided approach to tuning the optoelectronic properties of DNA-SWCNT complexes through DNA mutation. Applying this technique over two rounds of DNA mutagenesis and screening, we evolved a DNA-SWCNT sensor with a 57% enhancement in fluorescence intensity. Furthermore, this sensor shows retained selectivity and sensitivity towards the analyte of interest. Though we demonstrate this approach to improve sensor brightness, directed evolution offers far-reaching possibilities in the field that can also be applied to improve additional sensor properties such as sensitivity and selectivity.