Characteristics and outcomes of COVID-19 patients during B.1.1.529 (Omicron) dominance compared to B.1.617.2 (Delta) in 89 German hospitals.

BACKGROUND: The SARS-CoV-2 variant B.1.1.529 (Omicron) was first described in November 2021 and became the dominant variant worldwide. Existing data suggests a reduced disease severity with Omicron infections in comparison to B.1.617.2 (Delta). Differences in characteristics and in-hospital outcomes of COVID-19 patients in Germany during the Omicron period compared to Delta are not thoroughly studied. ICD-10-code-based severe acute respiratory infections (SARI) surveillance represents an integral part of infectious disease control in Germany. METHODS: Administrative data from 89 German Helios hospitals was retrospectively analysed. Laboratory-confirmed SARS-CoV-2 infections were identified by ICD-10-code U07.1 and SARI cases by ICD-10-codes J09-J22. COVID-19 cases were stratified by concomitant SARI. A nine-week observational period between December 6, 2021 and February 6, 2022 was defined and divided into three phases with respect to the dominating virus variant (Delta, Delta to Omicron transition, Omicron). Regression analyses adjusted for age, gender and Elixhauser comorbidities were applied to assess in-hospital patient outcomes. RESULTS: A total cohort of 4,494 inpatients was analysed. Patients in the Omicron dominance period were younger (mean age 47.8 vs. 61.6; p < 0.01), more likely to be female (54.7% vs. 47.5%; p < 0.01) and characterized by a lower comorbidity burden (mean Elixhauser comorbidity index 5.4 vs. 8.2; p < 0.01). Comparing Delta and Omicron periods, patients were at significantly lower risk for intensive care treatment (adjusted odds ratio 0.72 [0.57-0.91]; p = 0.005), mechanical ventilation (adjusted odds ratio 0.42 [0.31-0.57]; p < 0.001), and in-hospital mortality (adjusted odds ratio 0.42 [0.32-0.56]; p < 0.001). This also applied mostly to the separate COVID-SARI group. During the Delta to Omicron transition, case numbers of COVID-19 without SARI exceeded COVID-SARI for the first time in the pandemic's course. CONCLUSION: Patient characteristics and outcomes differ during the Omicron dominance period as compared to Delta suggesting a reduced disease severity with Omicron infections. SARI surveillance might play a crucial role in assessing disease severity of future SARS-CoV-2 variants.

The effect of tafamidis on the QTc interval in healthy subjects.

AIMS: The transthyretin (TTR) stabilizer, tafamidis, has demonstrated efficacy and safety in the treatment of TTR familial amyloid polyneuropathy (20 mg day(-1) ). Tafamidis use in TTR cardiomyopathy led to the study of the potential effect of tafamidis on the QTc interval in healthy subjects. METHODS: This randomized, three treatment, three period, six sequence crossover study with placebo, a positive control (moxifloxacin 400 mg) and tafamidis (400 mg, to achieve a supra-therapeutic Cmax of ~20 µg ml(-1) ) was conducted in healthy volunteers at three clinical research units. Oral dosing in each of the three treatment periods was separated by a washout period of ≥ 14 days. Serial triplicate 12-lead electrocardiograms were performed. QTc intervals were derived using the Fridericia correction method. Safety and tolerability were assessed by physical examination, vital signs measurement, laboratory analyses and monitoring of adverse events (AEs). RESULTS: A total of 42 subjects completed the study. The upper limit of the two-sided 90% confidence intervals (CIs) for the difference in baseline-adjusted QTc F between tafamidis 400 mg and placebo was <10 ms (non-inferiority criterion) for all time points. The lower limit of the two-sided 90% CI between moxifloxacin 400 mg and placebo exceeded 5 ms at the pre-specified moxifloxacin tmax of 3 h post-dose, confirming assay sensitivity. Cmax and AUC(0,24 h) for tafamidis were 20.36 µg ml(-1) and 305.4 µg ml(-1) h, respectively. There were no serious/severe AEs or treatment discontinuations due to AEs. CONCLUSIONS: This thorough QTc study suggests that a supra-therapeutic single 400 mg oral dose of tafamidis does not prolong the QTc interval and is well-tolerated in healthy volunteers.

The morphology of silver nanoparticles prepared by enzyme-induced reduction.

Silver nanoparticles were synthesized by an enzyme-induced growth process on solid substrates. In order to customize the enzymatically grown nanoparticles (EGNP) for analytical applications in biomolecular research, a detailed study was carried out concerning the time evolution of the formation of the silver nanoparticles, their morphology, and their chemical composition. Therefore, silver-nanoparticle films of different densities were investigated by using scanning as well as transmission electron microscopy to examine their structure. Cross sections of silver nanoparticles, prepared for analysis by transmission electron microscopy were additionally studied by energy-dispersive X-ray spectroscopy in order to probe their chemical composition. The surface coverage of substrates with silver nanoparticles and the maximum particle height were determined by Rutherford backscattering spectroscopy. Variations in the silver-nanoparticle films depending on the conditions during synthesis were observed. After an initial growth state the silver nanoparticles exhibit the so-called desert-rose or nanoflower-like structure. This complex nanoparticle structure is in clear contrast to the auto-catalytically grown spherical particles, which maintain their overall geometrical appearance while increasing their diameter. It is shown, that the desert-rose-like silver nanoparticles consist of single-crystalline plates of pure silver. The surface-enhanced Raman spectroscopic (SERS) activity of the EGNP structures is promising due to the exceptionally rough surface structure of the silver nanoparticles. SERS measurements of the vitamin riboflavin incubated on the silver nanoparticles are shown as an exemplary application for quantitative analysis.

Towards multiple readout application of plasmonic arrays.

In order to combine the advantages of fluorescence and surface-enhanced Raman spectroscopy (SERS) on the same chip platform, a nanostructured gold surface with a unique design, allowing both the sensitive detection of fluorescence light together with the specific Raman fingerprint of the fluorescent molecules, was established. This task requires the fabrication of plasmonic arrays that permit the binding of molecules of interest at different distances from the metallic surface. The most efficient SERS enhancement is achieved for molecules directly adsorbed on the metallic surface due to the strong field enhancement, but where, however, the fluorescence is quenched most efficiently. Furthermore, the fluorescence can be enhanced efficiently by careful adjustment of the optical behavior of the plasmonic arrays. In this article, the simultaneous application of SERS and fluorescence, through the use of various gold nanostructured arrays, is demonstrated by the realization of a DNA detection scheme. The results shown open the way to more flexible use of plasmonic arrays in bioanalytics.

Biofunctionalization of zinc oxide nanowires for DNA sensory applications.

We report on the biofunctionalization of zinc oxide nanowires for the attachment of DNA target molecules on the nanowire surface. With the organosilane glycidyloxypropyltrimethoxysilane acting as a bifunctional linker, amino-modified capture molecule oligonucleotides have been immobilized on the nanowire surface. The dye-marked DNA molecules were detected via fluorescence microscopy, and our results reveal a successful attachment of DNA capture molecules onto the nanowire surface. The electrical field effect induced by the negatively charged attached DNA molecules should be able to control the electrical properties of the nanowires and gives way to a ZnO nanowire-based biosensing device.

Development of a lab-on-a-chip device for diagnosis of plant pathogens.

A lab-on-a-chip system for rapid nucleic acid-based analysis was developed that can be applied for diagnosis of selected Phytophthora species as a first example for use in plant pathology. All necessary polymerase chain reaction process (PCR) and hybridization steps can be performed consecutively within a single chip consisting of two components, an inflexible and a flexible one, with integrated microchannels and microchambers. Data from the microarray is collected from a simple electrical measurement that is based on elementary silver deposition by enzymatical catalyzation. Temperatures in the PCR and in the hybridization zone are managed by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply for use on site. The specificity of the lab-on-a-chip system could be demonstrated for the tested five Phytophthora species. The two Pythium species gave signals below the threshold. The results of the electrical detection of the microarray correspond to the values obtained with the control method (optical grey scale analysis).

Lambda exonuclease pre-treatment for improved DNA-chip performance depends on the relative probe-target position.

The hybridisation characteristics of DNA targets to solid phase bound probes, e.g. in DNA microarrays, depend on the probe-target position and on target renaturation if a dsDNA target is used. We investigated a lambda exonuclease treatment of a PCR amplified dsDNA target to produce ssDNA with regard to probe-target position, treatment duration and inactivation time towards its impact on fluorescence or electrical signals on two DNA-chip formats. Surprisingly, the achieved amplification factors varied by three orders of magnitude, i.e. 2-1074 fold signal enhancement, depending on the relative probe-target position and readout scheme. The presented results can be used to design future studies involving lambda exonuclease preanalytic treatments.

Investigation on the second part of the electromagnetic SERS enhancement and resulting fabrication strategies of anisotropic plasmonic arrays.

In general, the electromagnetic mechanism is understood as the strongest contribution to the overall surface-enhanced Raman spectroscopy (SERS) enhancement. Due to the excitation of surface plasmons, a strong electromagnetic field is induced at the interfaces of a metallic nanoparticle leading to a drastic enhancement of the Raman scattering cross-section. Furthermore, the Raman scattered light expierences an emission enhancement due to the plasmon resonances of the nanoantennas. Herein, this second part of the electromagnetic enhancement phenomenon is investigated for different Raman bands of crystal violet by utilizing the anisotropic plasmonic character of gold nanorhomb SERS arrays. We aim at evaluating the effects of localized and propagating surface plasmon polariton modes as well as their combination on the scattered SERS intensity. From that point of view, design and fabrication strategies towards the fabrication of SERS arrays for excitation wavelengths in the visible and near-infrared (NIR) spectral region can be given, also using a double-resonant electromagnetic enhancement.

SERS as tool for the analysis of DNA-chips in a microfluidic platform.

A sequence-specific detection method of DNA is presented combining a solid chip surface for immobilisation of capture DNAs with a microfluidic platform and a readout of the chip based on SERS. The solid chip surface is used for immobilisation of different capture DNAs, where target strands can be hybridised and unbound surfactants can be washed away. For the detection via SERS, short-labelled oligonucleotides are hybridised to the target strands. This technique is combined with a microfluidic platform that enables a fast and automated preparation process. By applying a chip format, the problems of sequence-specific DNA detection in solution phase by means of SERS can be overcome. With this setup, we are able to distinguish between different complementary and non-complementary target sequences in one sample solution.

Novel bottom-up SERS substrates for quantitative and parallelized analytics.

Surface-enhanced Raman spectroscopy (SERS) is an emerging technology in the field of analytics. Due to the high sensitivity in connection with specific Raman molecular fingerprint information SERS can be used in a variety of analytical, bioanalytical, and biosensing applications. However, for the SERS effect substrates with metal nanostructures are needed. The broad application of this technology is greatly hampered by the lack of reliable and reproducible substrates. Usually the activity of a given substrate has to be determined by time-consuming experiments such as calibration or ultramicroscopic studies. To use SERS as a standard analytical tool, cheap and reproducible substrates are required, preferably with a characterization technique that does not interfere with the subsequent measurements. Herein we introduce an innovative approach to produce low-cost and large-scale reproducible substrates for SERS applications, which allows easy and economical production of micropatterned SERS active surfaces on a large scale. This approach is based on an enzyme-induced growth of silver nanostructures. The special structural feature of the enzymatically deposited silver nanoparticles prevents the breakdown of SERS activity even at high particle densities (particle density >60%) that lead to a conductive layer. In contrast to other approaches, this substrate exhibits a relationship between electrical conductivity and the resulting SERS activity of a given spot. This enables the prediction of the SERS activity of the nanostructure ensemble and therewith the controllable and reproducible production of SERS substrates of enzymatic silver nanoparticles on a large scale, utilizing a simple measurement of the electrical conductivity. Furthermore, through a correlation between the conductivity and the SERS activity of the substrates it is possible to quantify SERS measurements with these substrates.

UV cross-linking of unmodified DNA on glass surfaces.

The performance of DNA microarrays strongly depends on their surface properties. Furthermore, the immobilization method of the capture molecules is of importance for the efficiency of the microarray in terms of sensitivity and specificity. This work describes the immobilization of single-stranded capture oligonucleotides by UV cross-linking on silanated (amino and epoxy) glass surfaces. Thereby we used amino (NH2) and poly thymine/poly cytosine modifications of the capture sequences as well as unmodified capture molecules. The results were compared to UV cross-linking of the same DNA oligonucleotides on unmodified glass surfaces. Immobilization and hybridization efficiency was demonstrated by fluorescence and enzyme-induced deposition of silver nanoparticles. We found out that single-stranded DNA molecules do not require a special modification to immobilize them by UV cross-linking on epoxy- or amino-modified glass surfaces. However, higher binding rates can be achieved when using amino-modified oligonucleotides on an epoxy surface. The limit of detection for the used settings was 5 pM.

A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA.

Requirements for a point-of-care device are an easy and robust read-out and--above all--a simple handling. We integrated an established robust electrical read-out for DNA-chips into a microfluidic device, thereby creating an automated analysis system that combines the necessary steps for a chip-based analysis. It is based on the electrical detection of biotin-labeled DNA in a gap between two microstructured electrodes on the surface of a DNA-chip. The biotin serves as binding molecule for streptavidin-conjugated horseradish peroxidase. A following enzyme-induced silver deposition bridges the gap by a conductive layer. The miniaturized chip gives the possibility to realize a durable system suitable for point-of-care applications. To enable an initial automation, all corresponding process steps were executed in a miniaturized silicone flow cell. The required defined temperatures for the hybridization and the washing steps can be adjusted by a heating foil. This paper characterizes the performance of the flow cell based system in terms of reaction speed and analysis time, sensitivity as well as specificity, and the comparison to a conventional system, without flow cell. These first steps of automation and integration will help to realize a laboratory-independent bioanalytical tool, for the use outside of specialized laboratories for fast analysis of different chemical and biological applications.

Ultrafast plasmon dynamics and evanescent field distribution of reproducible surface-enhanced Raman-scattering substrates.

Surface-enhanced Raman scattering (SERS) is a potent tool in bioanalytical science because the technique combines high sensitivity with molecular specificity. However, the widespread and routine use of SERS in quantitative biomedical diagnostics is limited by tight requirements on the reproducibility of the noble metal substrates used. To solve this problem, we recently introduced a novel approach to reproducible SERS substrates. In this contribution, we apply ultrafast time-resolved spectroscopy to investigate the photo-induced collective charge-carrier dynamics in such substrates, which represents the fundamental origin of the SERS mechanism. The ultrafast experiments are accompanied by scanning-near field optical microscopy and SERS experiments to correlate the appearance of plasmon dynamics with the resultant evanescent field distribution and the analytically relevant SERS enhancement.

Flexible biochips for detection of biomolecules.

Miniaturization of biosensors is envisaged by the development of biochips consisting of parallel microarray patterns of binding sites on rigid substrates, such as glass or silicon. Thin plastic substrates are promising flexible alternatives because of the possibility for large-area roll-to-roll manufacturing of disposable chips at lower costs. Mature optical lithography technology faces many challenges when used to pattern flexible foils as a result of the substrate instabilities, especially at higher temperatures. In this work, flexible biochips with gold electrode patterns were fabricated on thin polyethylene naphthalate (PEN) foils using photolithography. The gold electrode structures of the chips were manufactured by direct metal patterning and by lift-off processing. Both methodologies resulted in well-defined electrode patterns as concluded from optical microscopy and scanning electron microscopy (SEM) characterization and resistance measurements. The biochips were successfully employed for the electrical and optical detection of DNA molecules. The DNA detection was based on the immobilization of capture DNA between electrode gaps, hybridization with biotin-labeled target DNA, and enzymatic silver enhancement.

Screen printing as cost-efficient fabrication method for DNA-chips with electrical readout for detection of viral DNA.

The fast development in the field of DNA analytics is driven by the need for cost-effective and high-throughput methods for the detection of biomolecules. The detection of DNA using metal nanoparticles as labels is an interesting alternative to the standard fluorescence technique. Fluorescence is highly sensitive and broadly established, but shows limitations, for example instability of the signal and the requirement for sophisticated and high-cost equipment. A recently developed approach realizes a method for the electrical detection of DNA, based on the induction of silver nanoparticles growth in microelectrode gaps on the surface of a DNA-chip. This breakthrough towards robust and cost-effective detection was still hampered by the need for microstructured (and therefore expensive) substrates. We demonstrate that it is possible to utilize screen printed electrode structures for a chip-based electrical DNA detection. The electrode structures were produced on a glass substrate which made an additional optical readout possible. The screen printed structures show the required precision and are compatible with the applied biochemical protocols. A comparison with chip substrates produced by standard photolithography showed the same sensitivity and specificity for the screen printed chips. Screen printing of electrode structures for DNA-chip with electrical detection offers an interesting and cost-efficient possibility to produce DNA-chips with microstructured electrodes.

DNA detection using a triple readout optical/AFM/MALDI planar microwell plastic chip.

A ready-to-spot disposable DNA chip for specific and sensitive detection of DNA was developed. Plastic copolymeric substrate chemistry was optimized to selectively couple the target DNA with the active chip surface. At the same time, the developed substrate limits the unspecific adsorption of probe DNA molecules or additional polar contaminants in the test samples to the chip surface. The combination of glycidyl and n-butyl methacrylates was found to best fit the requirements of the assay. The fabricated DNA microarrays have mechanical properties similar to those of the glass or silicon substrates and, at the same time, provide chemically reactive surfaces that do not require lengthy chemical modification. An additional advantage of the plastic microchip is its compatibility with different analytical readout techniques, such as mass spectrometry (MALDI-TOF/MS), optical detection (fluorescence and enzyme-induced metal deposition), and imaging techniques (atomic force microscopy). These multiple readout techniques have given us the ability to compare the sensitivity, selectivity, and robustness of current state-of-the-art bioanalytical methods on the same platform exemplified by successful DNA-based detection of human cytomegalovirus. The obtained sensitivity for enzymatically enhanced silver deposition (10(-15) M) surpasses that of conventional fluorescence readouts. In addition, the assay's dynamic range (10(-6)-10(-15) M), reproducibility, and reliability of the DNA probe detection speaks for the silver deposition method. At compromised sensitivity (10(-9) M), the length of the DNA probes could be checked and, alternatively, DNA single point polymorphisms could be analyzed.

Probing innovative microfabricated substrates for their reproducible SERS activity.

New types of microfabricated surface-enhanced Raman spectroscopy (SERS) active substrates produced by electron beam lithography and ion beam etching are introduced. In order to achieve large enhancement factors by using the lightning rod effect, we prepare arrays consisting of sharp-edged nanostructures instead of the commonly used dots. Two experimental methods are used for fabrication: a one-stage process, leading to gold nanostar arrays and a two-stage process, leading to gold nanodiamond arrays. Our preparation process guarantees high reproducibility. The substrates contain a number of arrays for practical applications, each 200x200 microm2 in size. To test the SERS activity of these nanostar and nanodiamond arrays, a monolayer of the dye crystal violet is used. Enhancement factors are estimated to be at least 130 for the nanodiamond and 310 for the nanostar arrays.

Microarray-based detection of dye-labeled DNA by SERRS using particles formed by enzymatic silver deposition.

The growing interest in DNA diagnostics is addressed today by microarrays with fluoresence detection. In our approach, we utilize spatially defined arrays of short oligonucleotides on a modified glass surface. Surface enhanced resonance Raman scattering (SERRS) is used to obtain molecularly specific spectra of the Raman-active dye-labeled DNA. Nanoparticles produced by enzymatic silver deposition are used as SERS-active substrate. They grow directly on the modified oligonucleotides and only in the spatially defined areas on the chip. Furthermore, they potentially offer several advantages for SERS detection. The nanoparticles are characterized and their ability for use as SERS- and SERRS-active substrate is estimated. Three different Raman-active dyes are investigated for their potential for involvement in sequence specific DNA analysis.