Development of a High-Throughput Mass Spectrometry-Based SARS-CoV-2 Immunoassay.

The serious impact of the Covid-19 pandemic underscores the need for rapid, reliable, and high-throughput diagnosis methods for infection. Current analytical methods, either point-of-care or centralized detection, are not able to satisfy the requirements of patient-friendly testing, high demand, and reliability of results. Here, we propose a two-point separation on-demand diagnostic strategy that uses laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) and adopts a stable yet cleavable ionic probe as a mass reporter. The use of this reporter enables ultrasensitive, interruptible, storable, restorable, and high-throughput on-demand detection. We describe a demonstration of the concept whereby we (i) design and synthesize a laser-cleavable reporter (DTPA), (ii) conjugate the reporter onto an antibody and verify the function of the conjugate, (iii) detect with good turnaround and high sensitivity the conjugated reporter, (iv) analyze quantitatively by using a laser-cleavable internal standard, and (v) identify negative and positive samples containing the spike protein. The protocol has excellent sensitivity (amol for the SARS-CoV-2 Spike S1 subunit antibody) without any amplification. This strategy is also applicable for the detection of other disease antigens besides SARS-CoV-2.

Metalation/Demetalation as a Postgelation Strategy To Tune the Mechanical Properties of Catenane-Crosslinked Gels.

Mechanically interlocked molecules (MIMs) possess unique architectures and nontraditional degrees of freedom that arise from well-defined topologies that are achieved through precise mechanical bonding. Incorporation of MIMs into materials can thus provide an avenue to discover new and emergent macroscale properties. Here, the synthesis of a phenanthroline-based [2]catenane crosslinker and its incorporation into polyacrylate organogels are described. Specifically, Cu(I) metalation and demetalation was used as a postgelation strategy to tune the mechanical properties of a gel by controlling the conformational motions of integrated MIMs. The organogels were prepared via thermally initiated free radical polymerization, and Cu(I) metal was added in MeOH to the pretreated, swollen gels. Demetalation of the gels was achieved by adding lithium cyanide and washing the gels. Changes in Young's and shear moduli, as well as tensile strength, were quantified through oscillatory shear rheology and tensile testing. The reported approach provides a general method for postgelation tuning of mechanical properties using metals and well-defined catenane topologies as part of a gel network architecture.

Iterative step-growth synthesis and degradation of unimolecular polyviologens under mild conditions.

An iterative step-growth addition method was used to expedite the gram-scale synthesis of main-chain polyviologens by several days, while also producing the longest main-chain polyviologen (i.e., 26 viologen subunits) reported to date. Facile degradation using inorganic and organic aqueous bases was also demonstrated for a representative oligoviologen (6V-Me·12Cl), a polyviologen (26V-Me·52Cl), and oligoviologen-crosslinked hydrogels.

Photoinduced Electron Transfer and Changes in Surface Free Energy in Polythiophene-Polyviologen Bilayered Thin Films.

Bipyridiniums, also known as viologens, are well-documented electron acceptors that are generally easy to synthesize on a large scale and reversibly cycle between three oxidation states (V2+, V•+, and V0). Accordingly, they have been explored in a number of applications that capitalize on their dynamic redox chemistry, such as redox-flow batteries and electrochromic devices. Viologens are also particularly useful in photoinduced electron transfer (PET) processes and therefore are of interest in photovoltaic applications that typically rely on electron-rich donors like polythiophene (PTh). However, the PET mechanism and relaxation dynamics between interfacing PTh and viologen-based thin films has not been well studied as a function of thickness of the acceptor layer. Here, a novel, bilayered thin film composite was fabricated by first spin-coating PTh onto glass slides, followed by spin-coating and curing polyviologen (PV)-based micron-sized films of variable thicknesses (0.5-11.3 µm) on top of the PTh layer. The electron-transfer mechanism and relaxation dynamics from the PTh sublayer into the upper PV film were investigated using femtosecond transient absorption (fTA) spectroscopy and electrochemistry to better understand how the charge-transfer/relaxation lifetimes could be extended using thicker PV acceptor films. The fTA experiments were performed under inert N2 conditions as well as in ambient O2. The latter shortened the lifetimes of the electrons in the PV layer, presumably due to O2 triplet-based trap sites. Contact angle measurements using H2O and MeI were also performed on top of the bilayered films to measure changes in surface free energy that would aid the assessment related to efficiency of the combined processes involving light penetration, photoexcitation, electron mobility, and relaxation from within the bilayered thin films. Insights gained from this work will support the development of future devices that employ viologen-based materials as an alternative electron-acceptor that is both easily processable and scalable.

Topologically Controlled Syntheses of Unimolecular Oligo[n]catenanes.

Catenanes are a well-known class of mechanically interlocked molecules that possess chain-like architectures and have been investigated for decades as molecular machines and switches. However, the synthesis of higher-order catenanes with multiple, linearly interlocked molecular rings has been greatly impeded by the generation of unwanted oligomeric byproducts and figure-of-eight topologies that compete with productive ring closings. Here, we report two general strategies for the synthesis of oligo[n]catenanes that rely on a molecular "zip-tie" strategy, where the "zip-tie" is a central core macrocycle precursor bearing two phenanthroline (phen) ligands to make odd-numbered oligo[n]catenanes, or a preformed asymmetric iron(II) complex consisting of two macrocycle precursors bearing phen and terpyridine ligands to make even-numbered oligo[n]catenanes. In either case, preformed macrocycles or [2]catenanes are threaded onto the central "zip-tie" core using metal templation prior to ring-closing metathesis (RCM) reactions that generate several mechanical bonds in one pot. Using these synthetic strategies, a family of well-defined linear oligo[n]catenanes were synthesized, where n = 2, 3, 4, 5, or 6 interlocked molecular rings, and n = 6 represents the highest number of linearly interlocked rings reported to date for any isolated unimolecular oligo[n]catenane.

One-Pot Synthesis of a Linear [4]Catenate Using Orthogonal Metal Templation and Ring-Closing Metathesis.

The efficient synthesis of well-defined, linear oligocatenanes possessing multiple mechanical bonds remains a formidable challenge in the field of mechanically interlocked molecules. Here, a one-pot synthetic strategy is described to prepare a linear [4]catenate using orthogonal metal templation between a macrocycle precursor, composed of terpyridine and phenanthroline ligands spaced by flexible glycol linkers, and a closed phenanthroline-based molecular ring. Implementation of two simultaneous ring-closing metathesis reactions after metal complexation resulted in the formation of three mechanical bonds. The linear [4]catenate product was isolated in 55% yield as a mixture of topological diastereomers. The intermediate metal complexes and corresponding interlocked products (with and without metals) were characterized by nuclear magnetic resonance, mass spectrometry, gel permeation chromatography, and UV-vis absorption spectroscopy. We envision that this general synthetic strategy may pave the way for the synthesis of higher order linear oligocatenates/catenanes with precise molecular weights and four or more interlocking molecular rings.

Dynamic, multimodal hydrogel actuators using porphyrin-based visible light photoredox catalysis in a thermoresponsive polymer network.

Hydrogels that can respond to multiple external stimuli represent the next generation of advanced functional biomaterials. Here, a series of multimodal hydrogels were synthesized that can contract and expand reversibly over several cycles while changing their mechanical properties in response to blue and red light, as well as heat (∼50 °C). The light-responsive behavior was achieved through a photoredox-based mechanism consisting of photoinduced electron transfer from a zinc porphyrin photocatalyst in its excited state to oligoviologen-based macrocrosslinkers, both of which were integrated into the hydrogel polymer network during gel formation. Orthogonal thermoresponsive properties were also realized by introducing N-isopropyl acrylamide (NIPAM) monomer simultaneously with hydroxyethyl acrylate (HEA) in the pre-gel mixture to produce a statistical 60 : 40 HEA : NIPAM polymer network. The resultant hydrogel actuators - crosslinked with either a styrenated viologen dimer (2V4+-St) or hexamer (6V12+-St) - were exposed to red or blue light, or heat, for up to 5 h, and their rate of contraction, as well as the corresponding changes in their physical properties (i.e., stiffness, tensile strength, Young's modulus, etc.), were measured. The combined application of blue light and heat to the 6V12+-St-based hydrogels was also demonstrated, resulting in hydrogels with more than two-fold faster contraction kinetics and dramatically enhanced mechanical robustness when fully contracted. We envision that the reported materials and the corresponding methods of remotely manipulating the dynamic hydrogels may serve as a useful blueprint for future adaptive materials used in biomedical applications.

Reversible Hydrogel Photopatterning: Spatial and Temporal Control over Gel Mechanical Properties Using Visible Light Photoredox Catalysis.

There is a growing interest in being able to control the mechanical properties of hydrogels for applications in materials, medicine, and biology. Primarily, changes in the hydrogel's physical properties, i.e., stiffness, toughness, etc., are achieved by modulating the network cross-linking chemistry. Common cross-linking strategies rely on (i) irreversible network bond degradation and reformation in response to an external stimulus, (ii) using dynamic covalent chemistry, or (iii) isomerization of integrated functional groups (e.g., azobenzene or spiropyran). Many of these strategies are executed using ultraviolet or visible light since the incident photons serve as an external stimulus that affords spatial and temporal control over the mechanical adaptation process. Here, we describe a different type of hydrogel cross-linking strategy that uses a redox-responsive cross-linker, incorporated in poly(hydroxyethyl acrylate)-based hydrogels at three different weight percent loadings, which consists of two viologen subunits tethered by hexaethylene glycol and capped with styrene groups at each terminus. These dicationic viologen subunits (V2+) can be reduced to their monoradical cations (V•+) through a photoinduced electron transfer (PET) process using a visible light-absorbing photocatalyst (tris(bipyridine)ruthenium(II) dichloride) embedded in the hydrogel, resulting in the intramolecular stacking of viologen radical cations, through radical-radical pairing interactions, while losing two positive charges and the corresponding counteranions from the hydrogel. It is shown how this concerted process ultimately leads to collapse of the hydrogel network and significantly (p < 0.05) increases (by nearly a factor of 2) the soft material's stiffness, tensile strength, and percent elongation at break, all of which is easily reversed via oxidation of the viologen subunits and swelling in water. Application of this reversible PET process was demonstrated by photopatterning the same hydrogel multiple times, where the pattern was "erased" each time by turning off the blue light (∼450 nm) source and allowing for oxidation and reswelling in between patterning steps. The areas of the hydrogel that were masked exhibited lower (by 1-2 kPa) shear storage moduli (G') than the areas that were irradiated for 1.5 h. Moreover, because the viologen subunits in the functional cross-linker are electrochromic, it is possible to visualize the regions of the hydrogel that undergo changes in mechanical properties. This visualization process was illustrated by photopatterning a larger hydrogel (∼9.5 cm on its longest side) with a photomask in the design of an array of stars.

Photoredox-Based Actuation of an Artificial Molecular Muscle.

The use of light to actuate materials is advantageous because it represents a cost-effective and operationally straightforward way to introduce energy into a stimuli-responsive system. Common strategies for photoinduced actuation of materials typically rely on light irradiation to isomerize azobenzene or spiropyran derivatives, or to induce unidirectional rotation of molecular motors incorporated into a 3D polymer network. Although interest in photoredox catalysis has risen exponentially in the past decade, there are far fewer examples where photoinduced electron transfer (PET) processes are employed to actuate materials. Here, a novel mode of actuation in a series of redox-responsive hydrogels doped with a visible-light-absorbing ruthenium-based photocatalyst is reported. The hydrogels are composed primarily of polyethylene glycol and low molar concentrations of a unimolecular electroactive polyviologen that is activated through a PET mechanism. The rate and degree of contraction of the hydrogels are measured over several hours while irradiating with blue light. Likewise, the change in mechanical properties-determined through oscillatory shear rheology experiments-is assessed as a function of polyviologen concentration. Finally, an artificial molecular muscle is fabricated using the best-performing hydrogel composition, and its ability to perform work, while irradiated, is demonstrated by lifting a small weight.