Asymmetric Mach-Zehnder Interferometric Biosensing for Quantitative and Sensitive Multiplex Detection of Anti-SARS-CoV-2 Antibodies in Human Plasma.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has once more emphasized the urgent need for accurate and fast point-of-care (POC) diagnostics for outbreak control and prevention. The main challenge in the development of POC in vitro diagnostics (IVD) is to combine a short time to result with a high sensitivity, and to keep the testing cost-effective. In this respect, sensors based on photonic integrated circuits (PICs) may offer advantages as they have features such as a high analytical sensitivity, capability for multiplexing, ease of miniaturization, and the potential for high-volume manufacturing. One special type of PIC sensor is the asymmetric Mach-Zehnder Interferometer (aMZI), which is characterized by a high and tunable analytical sensitivity. The current work describes the application of an aMZI-based biosensor platform for sensitive and multiplex detection of anti-SARS-CoV-2 antibodies in human plasma samples using the spike protein (SP), the receptor-binding domain (RBD), and the nucleocapsid protein (NP) as target antigens. The results are in good agreement with several CE-IVD marked reference methods and demonstrate the potential of the aMZI biosensor technology for further development into a photonic IVD platform.

A Miniature Bio-Photonics Companion Diagnostics Platform for Reliable Cancer Treatment Monitoring in Blood Fluids.

In this paper, we present the development of a photonic biosensor device for cancer treatment monitoring as a complementary diagnostics tool. The proposed device combines multidisciplinary concepts from the photonic, nano-biochemical, micro-fluidic and reader/packaging platforms aiming to overcome limitations related to detection reliability, sensitivity, specificity, compactness and cost issues. The photonic sensor is based on an array of six asymmetric Mach Zender Interferometer (aMZI) waveguides on silicon nitride substrates and the sensing is performed by measuring the phase shift of the output signal, caused by the binding of the analyte on the functionalized aMZI surface. According to the morphological design of the waveguides, an improved sensitivity is achieved in comparison to the current technologies (<5000 nm/RIU). This platform is combined with a novel biofunctionalization methodology that involves material-selective surface chemistries and the high-resolution laser printing of biomaterials resulting in the development of an integrated photonics biosensor device that employs disposable microfluidics cartridges. The device is tested with cancer patient blood serum samples. The detection of periostin (POSTN) and transforming growth factor beta-induced protein (TGFBI), two circulating biomarkers overexpressed by cancer stem cells, is achieved in cancer patient serum with the use of the device.

PLL-Poly(HPMA) Bottlebrush-Based Antifouling Coatings: Three Grafting Routes.

In this work, we compare three routes to prepare antifouling coatings that consist of poly(l-lysine)-poly(N-(2-hydroxypropyl)methacrylamide) bottlebrushes. The poly(l-lysine) (PLL) backbone is self-assembled onto the surface by charged-based interactions between the lysine groups and the negatively charged silicon oxide surface, whereas the poly(N-(2-hydroxypropyl)methacrylamide) [poly(HPMA)] side chains, grown by reversible addition-fragmentation chain-transfer (RAFT) polymerization, provide antifouling properties to the surface. First, the PLL-poly(HPMA) coatings are synthesized in a bottom-up fashion through a grafting-from approach. In this route, the PLL is self-assembled onto a surface, after which a polymerization agent is immobilized, and finally HPMA is polymerized from the surface. In the second explored route, the PLL is modified in solution by a RAFT agent to create a macroinitiator. After self-assembly of this macroinitiator onto the surface, poly(HPMA) is polymerized from the surface by RAFT. In the third and last route, the whole PLL-poly(HPMA) bottlebrush is initially synthesized in solution. To this end, HPMA is polymerized from the macroinitiator in solution and the PLL-poly(HPMA) bottlebrush is then self-assembled onto the surface in just one step (grafting-to approach). Additionally, in this third route, we also design and synthesize a bottlebrush polymer with a PLL backbone and poly(HPMA) side chains, with the latter containing 5% carboxybetaine (CB) monomers that eventually allow for additional (bio)functionalization in solution or after surface immobilization. These three routes are evaluated in terms of ease of synthesis, scalability, ease of characterization, and a preliminary investigation of their antifouling performance. All three coating procedures result in coatings that show antifouling properties in single-protein antifouling tests. This method thus presents a new, simple, versatile, and highly scalable approach for the manufacturing of PLL-based bottlebrush coatings that can be synthesized partly or completely on the surface or in solution, depending on the desired production process and/or application.

Design, Synthesis, and Characterization of Fully Zwitterionic, Functionalized Dendrimers.

Dendrimers are interesting candidates for various applications because of the high level of control over their architecture, the presence of internal cavities, and the possibility for multivalent interactions. More specifically, zwitterionic dendrimers modified with an equal number of oppositely charged groups have found use in in vivo biomedical applications. However, the design and control over the synthesis of these dendrimers remains challenging, in particular with respect to achieving full modification of the dendrimer. In this work, we show the design and subsequent synthesis of dendrimers that are highly charged while having zero net charge, that is zwitterionic dendrimers that are potential candidates for biomedical applications. First, we designed and fully optimized the synthesis of charge-neutral carboxybetaine and sulfobetaine zwitterionic dendrimers. Following their synthesis, the various zwitterionic dendrimers were extensively characterized. In this study, we also report for the first time the use of X-ray photoelectron spectroscopy as an easy-to-use and quantitative tool for the compositional analysis of this type of macromolecules that can complement techniques such as nuclear magnetic resonance and gel permeation chromatography. Finally, we designed and synthesized zwitterionic dendrimers that contain a variable number of alkyne and azide groups that allow straightforward (bio)functionalization via click chemistry.

Direct Creation of Biopatterns via a Combination of Laser-Based Techniques and Click Chemistry.

In this paper, we present the immobilization of thiol-modified aptamers on alkyne- or alkene-terminated silicon nitride surfaces by laser-induced click chemistry reactions. The aptamers are printed onto the surface by laser-induced forward transfer (LIFT), which also induces the covalent bonding of the aptamers by thiol-ene or thiol-yne reactions that occur upon UV irradiation of the thiol-modified aptamers with ns laser pulses. This combination of LIFT and laser-induced click chemistry allows the creation of high-resolution patterns without the need for masks. Whereas the click chemistry already takes place during the printing process (single-step process) by the laser pulse used for the printing process, further irradiation of the LIFT-printed aptamers by laser pulses (two-step process) leads to a further increase in the immobilization efficiency.

Versatile (bio)functionalization of bromo-terminated phosphonate-modified porous aluminum oxide.

Porous aluminum oxide (PAO) is a nanoporous material used for various (bio)technological applications, and tailoring its surface properties via covalent modification is a way to expand and refine its application. Specific and complex chemical modification of the PAO surface requires a stepwise approach in which a secondary reaction on a stable initial modification is necessary to achieve the desired terminal molecular architecture and reactivity. We here show that the straightforward initial modification of the bare PAO surface with bromo-terminated phosphonic acid allows for the subsequent preparation of PAO with a wide scope of terminal reactive groups, making it suitable for (bio)functionalization. Starting from the initial bromo-terminated PAO, we prepared PAO surfaces presenting various terminal functional groups, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS). We also show that this wide scope of easily accessible tailored reactive PAO surfaces can be used for subsequent modification with (bio)molecules, including carbohydrate derivatives and fluorescently labeled proteins.

Covalent attachment of 1-alkenes to oxidized platinum surfaces.

We report the formation of covalently bound alkyl layers onto oxidized Pt (PtOx) substrates by reaction with 1-alkenes as a novel way to bind organic molecules to metal surfaces. The organic layers were characterized by static contact angle, infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The grafted alkyl layers display a hydrolytic stability that is comparable to that of alkyl thiols on Au. PtOx-alkene attachment is compatible with terminal ester moieties enabling further anchoring of functional groups, such as redox-active ferrocene, and thus has great potential to extend monolayer chemistry on noble metals.

Micropatterned ferrocenyl monolayers covalently bound to hydrogen-terminated silicon surfaces: effects of pattern size on the cyclic voltammetry and capacitance characteristics.

The effect of the size of patterns of micropatterned ferrocene (Fc)-functionalized, oxide-free n-type Si(111) surfaces was systematically investigated by electrochemical methods. Microcontact printing with amine-functionalized Fc derivatives was performed on a homogeneous acid fluoride-terminated alkenyl monolayer covalently bound to n-type H-terminated Si surfaces to give Fc patterns of different sizes (5 × 5, 10 × 10, and 20 × 20 µm(2)), followed by backfilling with n-butylamine. These Fc-micropatterned surfaces were characterized by static water contact angle measurements, ellipsometry, X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRRAS), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The charge-transfer process between the Fc-micropatterned and underlying Si interface was subsequently studied by cyclic voltammetry and capacitance. By electrochemical studies, it is evident that the smallest electroactive ferrocenyl patterns (i.e., 5 × 5 µm(2) squares) show ideal surface electrochemistry, which is characterized by narrow, perfectly symmetric, and intense cyclic voltammetry and capacitance peaks. In this respect, strategies are briefly discussed to further improve the development of photoswitchable charge storage microcells using the produced redox-active monolayers.

Covalent surface modification of oxide surfaces.

The modification of surfaces by the deposition of a robust overlayer provides an excellent handle with which to tune the properties of a bulk substrate to those of interest. Such control over the surface properties becomes increasingly important with the continuing efforts at down-sizing the active components in optoelectronic devices, and the corresponding increase in the surface area/volume ratio. Relevant properties to tune include the degree to which a surface is wetted by water or oil. Analogously, for biosensing applications there is an increasing interest in so-called "romantic surfaces": surfaces that repel all biological entities, apart from one, to which it binds strongly. Such systems require both long lasting and highly specific tuning of the surface properties. This Review presents one approach to obtain robust surface modifications of the surface of oxides, namely the covalent attachment of monolayers.

Ambient surface analysis of organic monolayers using direct analysis in real time Orbitrap mass spectrometry.

A better characterization of nanometer-thick organic layers (monolayers) as used for engineering surface properties, biosensing, nanomedicine, and smart materials will widen their application. The aim of this study was to develop direct analysis in real time high-resolution mass spectrometry (DART-HRMS) into a new and complementary analytical tool for characterizing organic monolayers. To assess the scope and formulate general interpretation rules, DART-HRMS was used to analyze a diverse set of monolayers having different chemistries (amides, esters, amines, acids, alcohols, alkanes, ethers, thioethers, polymers, sugars) on five different substrates (Si, Si3N4, glass, Al2O3, Au). The substrate did not play a major role except in the case of gold, for which breaking of the weak Au-S bond that tethers the monolayer to the surface, was observed. For monolayers with stronger covalent interfacial bonds, fragmentation around terminal groups was found. For ester and amide-terminated monolayers, in situ hydrolysis during DART resulted in the detection of ions characteristic of the terminal groups (alcohol, amine, carboxylic acid). For ether and thioether-terminated layers, scission of C-O or C-S bonds also led to the release of the terminal part of the monolayer in a predictable manner. Only the spectra of alkane monolayers could not be interpreted. DART-HRMS allowed for the analysis of and distinction between monolayers containing biologically relevant mono or disaccharides. Overall, DART-HRMS is a promising surface analysis technique that combines detailed structural information on nanomaterials and ultrathin films with fast analyses under ambient conditions.

Organic monolayers from 1-alkynes covalently attached to chromium nitride: alkyl and fluoroalkyl termination.

Strategies to modify chromium nitride (CrN) surfaces are important because of the increasing applications of these materials in various areas such as hybrid electronics, medical implants, diffusion barrier layers, corrosion inhibition, and wettability control. The present work presents the first surface immobilization of alkyl and perfluoro-alkyl (from C6 to C18) chains onto CrN substrates using appropriately functionalized 1-alkynes, yielding covalently bound, high-density organic monolayers with excellent hydrophobic properties and a high degree of short-range order. The obtained monolayers were characterized in detail by water contact angle, X-ray photoelectron spectroscopy (XPS), ellipsometry, and infrared reflection absorption spectroscopy (IRRAS).

Covalently attached organic monolayers onto silicon carbide from 1-alkynes: molecular structure and tribological properties.

In order to achieve improved tribological and wear properties at semiconductor interfaces, we have investigated the thermal grafting of both alkylated and fluorine-containing ((C(x)F(2x+1))-(CH2)n-) 1-alkynes and 1-alkenes onto silicon carbide (SiC). The resulting monolayers display static water contact angles up to 120°. The chemical composition of the covalently bound monolayers was studied by X-ray photoelectron spectroscopy (XPS), infrared reflection-absorption spectroscopy (IRRAS), and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. These techniques indicate the presence of acetal groups at the organic-inorganic interface of alkyne-modified SiC surfaces. The tribological properties of the resulting organic monolayers with fluorinated or nonfluorinated end groups were explored using atomic force microscopy (AFM). It was found that the fluorinated monolayers exhibit a significant reduction of adhesion forces, friction forces, and wear resistance compared with non-fluorinated molecular coatings and especially bare SiC substrates. The successful combination of hydrophobicity and excellent tribological properties makes these strongly bound, fluorinated monolayers promising candidates for application as a thin film coating in high-performance microelectronic devices.

Hexadecadienyl monolayers on hydrogen-terminated Si(111): faster monolayer formation and improved surface coverage using the enyne moiety.

To further improve the coverage of organic monolayers on hydrogen-terminated silicon (H-Si) surfaces with respect to the hitherto best agents (1-alkynes), it was hypothesized that enynes (H-C≡C-HC═CH-R) would be even better reagents for dense monolayer formation. To investigate whether the increased delocalization of ß-carbon radicals by the enyne functionality indeed lowers the activation barrier, the kinetics of monolayer formation by hexadec-3-en-1-yne and 1-hexadecyne on H-Si(111) were followed by studying partially incomplete monolayers. Ellipsometry and static contact angle measurements indeed showed a faster increase of layer thickness and hydrophobicity for the hexadec-3-en-1-yne-derived monolayers. This more rapid monolayer formation was supported by IRRAS and XPS measurements that for the enyne show a faster increase of the CH2 stretching bands and the amount of carbon at the surface (C/Si ratio), respectively. Monolayer formation at room temperature yielded plateau values for hexadec-3-en-1-yne and 1-hexadecyne after 8 and 16 h, respectively. Additional experiments were performed for 16 h at 80° to ensure full completion of the layers, which allows comparison of the quality of both layers. Ellipsometry thicknesses (2.0 nm) and contact angles (111-112°) indicated a high quality of both layers. XPS, in combination with DFT calculations, revealed terminal attachment of hexadec-3-en-1-yne to the H-Si surface, leading to dienyl monolayers. Moreover, analysis of the Si2p region showed no surface oxidation. Quantitative XPS measurements, obtained via rotating Si samples, showed a higher surface coverage for C16 dienyl layers than for C16 alkenyl layers (63% vs 59%). The dense packing of the layers was confirmed by IRRAS and NEXAFS results. Molecular mechanics simulations were undertaken to understand the differences in reactivity and surface coverage. Alkenyl layers show more favorable packing energies for surface coverages up to 50-55%. At higher coverages, this packing energy rises quickly, and there the dienyl packing becomes more favorable. When the binding energies are included the difference becomes more pronounced, and dense packing of dienyl layers becomes more favorable by 2-3 kcal/mol. These combined data show that enynes provide the highest-quality organic monolayers reported on H-Si up to now.

Molecular modeling of alkyl and alkenyl monolayers on hydrogen-terminated Si(111).

On H-Si(111) surfaces monolayer formation with 1-alkenes results in alkyl monolayers with a Si-C-C linkage, while 1-alkynes yield alkenyl monolayers with a Si-C═C linkage. Recently, considerable structural differences between both types of monolayers were observed, including an increased thickness, improved packing, and higher surface coverage for the alkenyl monolayers. The precise origin thereof could experimentally not be clarified yet. Therefore, octadecyl and octadecenyl monolayers on Si(111) were studied in detail by molecular modeling via PCFF molecular mechanics calculations on periodically repeated slabs of modified surfaces. After energy minimization the packing energies, structural properties, close contacts, and deformations of the Si surfaces of monolayers structures with various substitution percentages and substitution patterns were analyzed. For the octadecyl monolayers all data pointed to a substitution percentage close to 50-55%, which is due the size of the CH(2) groups near the Si surface. This agrees with literature and the experimentally determined coverage of octadecyl monolayers. For the octadecenyl monolayers the minimum in packing energy per chain is calculated around 60% coverage, i.e., close to the experimentally observed value of 65% [Scheres et al. Langmuir 2010, 26, 4790], and this packing energy is less dependent on the substitution percentage than calculated for alkyl layers. Analysis of the chain conformations, close contacts, and Si surface deformation clarifies this, since even at coverages above 60% a relatively low number of close contacts and a negligible deformation of the Si was observed. In order to evaluate the thermodynamic feasibility of the monolayer structures, we estimated the binding energies of 1-alkenes and 1-alkynes to the hydrogen-terminated Si surface at a range of surface coverages by composite high-quality G3 calculations and determined the total energy of monolayer formation by adding the packing energies and the binding energies. It was shown that due to the significantly larger reaction exothermicity of the 1-alkynes, thermodynamically even a substitution percentage as high as 75% is possible for octadecenyl chains. However, because sterically (based on the van der Waals footprint) a coverage of 69% is the maximum for alkyl and alkenyl monolayers, the optimal substitution percentage of octadecenyl monolayers will be presumably close to this latter value, and the experimentally observed 65% is likely close to what is experimentally maximally obtainable with alkenyl monolayers.

Light-enhanced microcontact printing of 1-alkynes onto hydrogen-terminated silicon.

A method for the direct patterning of 1-alkynes onto hydrogen-terminated silicon is presented. It combines microcontact printing with illumination through the stamp, and results in the formation of an alkenyl monolayer. The formation of heterogeneous monolayers is demonstrated by subsequent backfilling.

Micro- and nanopatterning of functional organic monolayers on oxide-free silicon by laser-induced photothermal desorption.

The photothermal laser patterning of functional organic monolayers, prepared on oxide-free hydrogen-terminated silicon, and subsequent backfilling of the laser-written lines with a second organic monolayer that differs in its terminal functionality, is described. Since the thermal monolayer decomposition process is highly nonlinear in the applied laser power density, subwavelength patterning of the organic monolayers is feasible. After photothermal laser patterning of hexadecenyl monolayers, the lines freed up by the laser are backfilled with functional acid fluoride monolayers. Coupling of cysteamine to the acid fluoride groups and subsequent attachment of Au nanoparticles allows easy characterization of the functional lines by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Depending on the laser power and writing speed, functional lines with widths between 1.1 µm and 250 nm can be created. In addition, trifluoroethyl-terminated (TFE) monolayers are also patterned. Subsequently, the decomposed lines are backfilled with a nonfunctional hexadecenyl monolayer, the TFE stripes are converted into thiol stripes, and then finally covered with Au nanoparticles. By reducing the lateral distance between the laser lines, Au-nanoparticle stripes with widths close to 100 nm are obtained. Finally, in view of the great potential of this type of monolayer in the field of biosensing, the ease of fabricating biofunctional patterns is demonstrated by covalent binding of fluorescently labeled oligo-DNA to acid-fluoride-backfilled laser lines, which--as shown by fluorescence microscopy--is accessible for hybridization.

Self-assembly of organic monolayers onto hydrogen-terminated silicon: 1-alkynes are better than 1-alkenes.

Recently, a new method for the preparation of high-quality organic monolayers with 1-alkynes at room temperature in the dark (i.e., without any external activation) was reported. To pinpoint the precise origin of this self-assembly process and to compare the reactivity of 1-alkenes and 1-alkynes toward hydrogen-terminated Si(111) [H-Si(111)], we followed the gradual formation of both monolayers at room temperature by static water contact angle measurements. Subsequently, attenuated total reflection infrared spectroscopy (ATR-IR) and X-ray photoelectron spectroscopy (XPS) were used to obtain detailed information about the structure and quality of the resulting monolayers. Our data clearly demonstrate that 1-alkynes are considerably more reactive toward H-Si(111) than 1-alkenes. 1-Alkynes are able to self-assemble into densely packed hydrophobic monolayers without any external activation (i.e., at room temperature under ambient light and even in the dark) whereas for 1-alkenes under the same conditions hardly any reactivity toward H-Si(111) was observed. The self-assembly of 1-alkynes on H-Si(111) at room temperature is explained by three factors: the higher nucleophilicity of 1-alkynes, which results in a facile attack at the electron-hole pairs at the H-Si surface and easy Si-C bond formation, the stabilization of the beta radical by delocalization over the double bond, and the lower-energy barrier encountered for H abstractions.