Frank F. Bier

Universität Potsdam · Institute of Biochemistry and Biology

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in t...

Around 30% of the scientific papers published on imprinted polymers describe the recognition of proteins, nucleic acids, viruses, and cells. The straightforward synthesis from only one up to six functional monomers and the simple integration into a sensor are significant advantages as compared with enzymes or antibodies. Furthermore, they can be sy...

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alte...

Correction for ‘Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein’ by Zsófia Bognár et al., Chem. Sci., 2022, 13, 1263–1269, https://doi.org/10.1039/D1SC04502D.

This study focuses on three key aspects: a) crude throat swab sample as 30 template for RT-LAMP reaction, b) biotinylated DNA probes enhanced specificity for LFA readout and c) digital semi-quantification of LFA readout. The throat swab samples from SARS-CoV-2 positive and negative patients have been used in their crude (no cleaning or pre-treatmen...

Multifunctional nanosized metal-organic frameworks (NMOFs) have advanced rapidly over the past decade to develop drug delivery systems (DDSs). These material systems still lack precise and selective cellular targeting, as well as the fast release of the quantity of drugs that are simply adsorbed within and on the external surface of nanocarriers, w...

Over the last few decades, cancer has been considered a clinical challenge, being among the leading causes of mortality all over the world. Although many treatment approaches have been developed for cancer, chemotherapy is still the most utilized in the clinical setting. However, the available chemotherapeutics-based treatments have several caveats...

Dielectrophoresis (DEP) is an AC electrokinetic effect that is proven to be effective for the immobilization of not only cells, but also of macromolecules, e.g., antibodies and enzyme molecules. In our previous work, we have already demonstrated the high catalytic activity of immobilized horseradish peroxidase after DEP. To evaluate the suitability...

Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a...

Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In...

Most people infected by the SARS-CoV-2 virus which causes COVID-19 disease experience mild or no symptoms. Severe forms of the disease are often marked by a hyper-inflammatory response known as a cytokine storm. Thus, biomarker tests which can identify these patients and place them on the appropriate treatment regime at the earliest possible phase...

DNA Nanoconstructs Electrically controllable DNA nanoconstructs are used to investigate the binding interaction between Influenza A viruses and a receptor peptide called PeB by Marlen Kruse and co‐workers in article number 2101141. Virus material is immobilized on gold electrodes, which allows to target virus‐proteins in their native environment.

The use of AC electrokinetic forces, like dielectrophoresis and AC electroosmosis, as a simple and fast method to immobilize sub‐micrometer objects onto nanoelectrode arrays is presented. Due to its medical relevance the influenza virus is chosen as a model organism. One of the outstanding features is that immobilization of viral material to the el...

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently...

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both...

Electrically controllable deoxyribonuclic acid (DNA) nanolevers are used to investigate the binding interaction between Influenza A/Aichi/2/1968 and the peptide called “PeB”, which specifically binds the viral surface protein hemagglutinin. PeB is immobilized on gold electrodes of a “switchSENSE” biochip by conjugation to DNA‐strands that are hybri...

In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of p...

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)‐2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross‐reactivity between SARS‐CoV‐2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of s...

Background: The ability to create nanostructures with biomolecules is one of the key elements in nanobiotechnology. One of the problems is the expensive and mostly custom made equipment which is needed for their development. We intended to reduce material costs and aimed at miniaturization of the necessary tools that are essential for nanofabricati...

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but may be a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development...

The application of inhomogeneous AC electric fields for molecular immobilization is a very fast and simple method that does not require any adaptions to the molecule’s functional groups or charges. Here, the method is applied to a completely new category of molecules: small organic fluorescence dyes, whose dimensions amount to only 1 nm or even les...

Diabetic foot ulcer (DFU) is a major complication of diabetes with highmorbidity and mortality rates. The pathogenesis of DFUs is governed by a complexmilieu of environmental and host factors. The empirical treatment is initially basedon wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time-con...

Chronic nonhealing diabetic foot ulcers (DFUs) are a serious complication of diabetes and are further exacerbated by bacterial colonization. The microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance, and virulence. T...

Most biochemical reactions depend on the pH value of the aqueous environment and some are strongly favoured to occur in an acidic environment. A non-invasive control of pH to tightly regulate such reactions with defined start and end points is a highly desirable feature in certain applications, but has proven difficult to achieve so far. We report...

In vitro cultured neuronal networks with defined connectivity are required to improve neuronal cell culture models. However, most protocols for their formation do not provide sufficient control of the direction and timing of neurite outgrowth with simultaneous access for analytical tools such as immunocytochemistry or patch-clamp recordings. Here w...

Many photochemical or photobiological applications require the use of high power ultraviolet light sources, such as high-pressure mercury arc lamps. In addition, many photo-induced chemical, biochemical and biological applications require either a combinatorial setting or a parallel assay of multiple samples under the same environmental conditions...

The spatial control of DNA and of self-assembled DNA constructs is a prerequisite for the preparation of DNA-based nanostructures and microstructures and a useful tool for studies on single DNA molecules. Here we describe a protocol for the accumulation of dissolved λ-DNA molecules between planar microelectrodes by the action of inhomogeneous radio...

Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and...

Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spect...

Lab-on-a-chip assays allow rapid analysis of one or more molecular analytes on an automated user-friendly platform. Here we describe a fully automated assay and readout for measurement of vitamin D levels in less than 15 min using the Fraunhofer in vitro diagnostics platform. Vitamin D (25-hydroxyvitamin D3 [25(OH)D3]) dilution series in buffer wer...

We demonstrate the immobilisation of polystyrene nanoparticles on vertical nano-electrodes by means of dielectrophoresis. The electrodes have diameters of 500 nm or 50 nm, respectively, and are arranged in arrays of several thousand electrodes, allowing many thousands of experiments in parallel. At a frequency of 15 kHz, which is found favourable f...

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers e...

Employing electric phenomena for the spatial manipulation of bioparticles from whole cells down to dissolved molecules has become a useful tool in biotechnology and analytics. AC electrokinetic effects like dielectrophoresis and AC electroosmosis are increasingly used to concentrate, separate and immobilize DNA and proteins. With the advance of pho...

Neisseria gonorrhoeae is the causative organism of gonorrhoea, a sexually transmitted disease that globally accounts for an estimated 80 to 100 million new infections per year. Increasing resistances to all common antibiotics used for N. gonorrhoeae treatment pose the risk of an untreatable disease. Further knowledge of ways of infection and host i...

Alanine scans of the identified epitopes of A) NGO0326_1_5, B) NGO0564_1_1, C) NGO0564_1_23, D) NGO0592_1_3, E) NGO0777_1_6. The boxes embody 50% of the values, while the whiskers comprise 98% of the data. Outliers are marked with a small x. Median values are indicated as a horizontal line, while the mean values are marked by a small square. Myelin...

Alanine scan of the identified epitope of NGO0170_1_3. The first and the last peptide are marked by a minus. The first peptide is additionally marked by a 1. The first fifteen spots correspond to the epitope sequence LLAREKIAAGATETI where each amino acid is substituted subsequently and each peptide is immobilised consecutively on one of the spots o...

Specificity controls of the identified peptides with epitope features of A) NGO0326, B) NGO0564, C) NGO0592, D) NGO0777, E) NGO1429, F) NGO1577. Almost all peptides showed specific binding for anti-N. gonorrhoeae antibodies. Only two peptides of NGO0592 (C) showed unspecific binding to the polyclonal anti-N. meningitidis antibody correlating to app...

Alanine scans of the identified epitopes of A) NGO1429_1_3, B) NGO1429_3_7, C) NGO1577_1_2, D) NGO1577_1_9, E) NGO1852_1_4. The boxes embody 50% of the values, while the whiskers comprise 98% of the data. Outliers are marked with a small x. Median values are indicated as a horizontal line, while the mean values are marked by a small square. Myelin...

Alanine scans of the identified epitopes of A) NGO2094_1_3c, B) NGO2094_1_10c, C) NGO2095_1_1, D) NGO2095_2_3c, E) NGO2095_2_13c, F) NGO2095_4_10. The boxes embody 50% of the values, while the whiskers comprise 98% of the data. Outliers are marked with a small x. Median values are indicated as a horizontal line, while the mean values are marked by...

Specificity controls of the identified peptides with epitope features of the remaining polypeptides. A) NGO1656, B) NGO1852, C) NGO2094, D) the first three potential epitopes NGO2095, E) further two potential epitopes NGO2095. Almost all peptides showed specific binding for anti-N. gonorrhoeae antibodies. Only two peptides of NGO2095 showed signal...

We identified Alizarin Red S and other well known fluorescent dyes useful for the online detection of pyrophosphate in enzymatic assays, including the loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) assays. An iterative screening was used for a selected set of compounds to first secure enzyme compatibility, evaluat...

Lab-on-a-chip assays allow rapid identification of multiple parameters on an automated user-friendly platform. Here we describe a fully automated multiplex immunoassay and readout in less than 15 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable inexpensive point-of-care profiling of sera or a single drop of blood from patients...

Lab-on-a-chip multiplex assays allow a rapid identification of multiple parameters in an automated manner. Here we describe a lab-based preparation followed by a rapid and fully automated DNA microarray hybridization and readout in less than 10 min using the Fraunhofer in vitro diagnostics (ivD) platform to enable rapid identification of bacterial...

Acute kidney injury (AKI) is a major health issue, affecting large numbers of patients worldwide. It is associated with an increase in complications and poor prognostics if diagnosis is delayed. Medical guidelines are routinely employed to classify different AKI stages, but guidance on the early detection of AKI risk is limited. In this paper, we p...

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy...

Substitutional analysis of peptide PeB using additional influenza strains. Labeled influenza California H1N1 (left), New York H3N2 (middle) and Victoria H3N2 (right) were used as analytes. Numbers represent mean value of the contrast relative to contrast for positive control fetuin. False-colors are used to illuminate fluorescence intensities, colo...

Analysis of peptide (PeB) structure. Solutions of PeB have been analyzed for structural features using circular dichroism (CD) spectroscopy (A) at physiological pH (7.4) and at pH 2, below the isoelectric point of the peptide. Further, the melting temperature profile of peptide PeB under both pH conditions was measured (B). (TIF)

Per-residue decomposition of binding free energy. Contributions of individual residues of PeB (black) and PeBGF (grey) to the binding free energies of the corresponding PeB-HA or PeBGF-HA complexes. A) Electrostatic, B) van der Waals and C) polar contributions to the total binding free energy change. The mutation of residue 6 (D6G) replacing a char...

SPR sensorgrams for Aichi H3N2 binding to peptides. Immobilized peptides were A) PeA-Lys (0.77 pmol mm-2), B) PeB-Lys (0.57 pmol mm-2) and C) PeC-Lys (1.93 pmol mm-2). Injection of virus was initiated at t = 69 s and terminated at t = 569 s. Numbers indicate virus injections at concentrations of up to 50 μg ml-1. (TIF)

Cell viability of MDCK II in the presence of peptides. MDCK II cells were treated 24 h with peptides PeB, PeBGF, and PeBP before cell viability was assessed by a MTS reagent. (TIF)

Neuraminidase activity assay. Enzyme activity in the presence of the inhibitor DANA (N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid) and PeB. Relative fluorescence of 4-Methylumbelliferone was measured after enzymatic conversion of MUNANA (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid) by neuraminidase following a known protocol [30]. Data show me...

Contributions of individual residues of HA to the binding free energies of the corresponding PeB-HA complexes. A) Wildtype HA (Aichi H3N2), and two single mutants B) T155I and C) T131I. (TIF)

Inhibition of binding of Aichi H3N2 viruses to α-2,6’-sialyllactose. Biotinylated α-2,6’-sialyllactose was immobilized on a neutravidin modified surface (650 RU). Virus was preincubated with peptide PeB or α-2,6’-sialyllactose before injection. Lines represent a sigmoidal fit model of the data (mean values of duplicate experiments). (TIF)

Proteins used for peptide binding specificity. (DOCX)

Alignment of HA sequences of used influenza viruses. Amino acids involved in binding of PeB as obtained from MD-simulations are highlighted in gray. PDB2vir: sequence obtained from protein database; A/mute/swan/R901/06-H7N1: sequence obtained from Prof. Harder (Friedrich-Loeffler-Institut, Riems, Germany); all other sequences were obtained from inf...

Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures a...

Schematic view of the protein primary sequence (yellow bar) from protein NGO0592 with annotated identified oligopeptides (brown bars). In total, three oligopeptides were identified; two of them overlapping between amino acids 79 and 148, the third in a different part of the protein spanning from amino acid residue 240 to 285. (TIF)

Amplification curves of the real-time PCR (n = 3). The Cp values were 27.25 ± 0.82 for NGO1634, 20.94 ± 1.7 for GDH (NGO0715) and > 35 for the controls. (TIF)

Summary of available homologous structures of all novel immunogenic proteins. Query coverage, E-values and sequence similarities obtained by PSI-BLAST (3 iterations) against the PDB database are listed. Only an excerpt of the complete list is shown containing the top hits and the template chosen by I-TASSER (highlighted in grey). For NGO0170 the ho...

Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still...

Protein molecules are aligned and immobilized from solution by AC electric fields. In a single-step experiment, the enhanced green fluorescent proteins are immobilized on the surface as well as at the edges of planar nanoelectrodes. Alignment is found to follow the molecules' geometrical shape with their longitudinal axes parallel to the electric f...

Herein we present an efficient synthesis of a biomimetic with modular construction that can be specifically bound by the mannose binding FimH protein – a surface adhesion protein of E. coli bacteria. The synthesis combines the new and interesting DBD dye with the carbohydrate ligand mannose via Click reaction. We demonstrate the binding to E. coli...

An antigen modified gold electrode based on a self-assembled lipoic acid monolayer has been developed. The contact angle measurement and cyclic voltammetry confirm the formation of a dense self-assembled monolayer on gold from 100 mM lipoic acid in ethanol. The electrochemical behavior shows a stable activity in a range of potential from -0.2 V to...

A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of t...

A straightforward synthesis strategy to multimerize a peptide mimotopes for antibody B13-DE1 recognition is described based on lysine dendrons as multivalent scaffolds. Lysine dendrons that possess N-terminal alkyne residues at the periphery were quantitative functionalized with azido peptides using click chemistry. The solid phase peptide synthesi...

The combination of alternating electric fields with nanometer-sized electrodes allows the permanent immobilization of proteins by dielectrophoretic force. Here, atomic force microscopy is introduced as a quantification method, and results are compared with fluorescence microscopy. Experimental parameters, e.g. the applied voltage and the duration o...

Background: Cardiovascular disease (CVD) is one of the major causes of death worldwide. Numerous genetic risk factors in lipid metabolism, including mutations of LDLR, APOB, and PCSK9, as well as polymorphisms of CETP and APOE, have been found to associate with CVD. Methods: In this study, a two-dye based arrayed primer extension (APEX) microarray...

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case β-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-es...

Para-maleimidophenyl (p-MP) modified gold surfaces have been prepared by one-step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modi...

Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently li...