Idiopathic pulmonary fibrosis (IPF): Diagnostic routes using novel biomarkers.

Idiopathic pulmonary fibrosis (IPF) diagnosis is still the diagnosis of exclusion. Differentiating from other forms of interstitial lung diseases (ILDs) is essential, given the various therapeutic approaches. The IPF course is now unpredictable for individual patients, although some genetic factors and several biomarkers have already been associated with various IPF prognoses. Since its early stages, IPF may be asymptomatic, leading to a delayed diagnosis. The present review critically examines the recent literature on molecular biomarkers potentially useful in IPF diagnostics. The examined biomarkers are grouped into breath and sputum biomarkers, serologically assessed extracellular matrix neoepitope markers, and oxidative stress biomarkers in lung tissue. Fibroblasts and complete blood count have also gained recent interest in that respect. Although several biomarker candidates have been profiled, there has yet to be a single biomarker that proved specific to the IPF disease. Nevertheless, various IPF biomarkers have been used in preclinical and clinical trials to verify their predictive and monitoring potential.

Molecularly imprinted polymer-based extended-gate field-effect transistor (EG-FET) chemosensor for selective determination of matrix metalloproteinase-1 (MMP-1) protein.

A conducting molecularly imprinted polymer (MIP) film was integrated with an extended-gate field-effect transistor (EG-FET) transducer to determine epitopes of matrix metalloproteinase-1 (MMP-1) protein biomarker of idiopathic pulmonary fibrosis (IPF) selectively. Most suitable epitopes for imprinting were selected with Basic Local Alignment Search Tool software. From a pool of MMP-1 epitopes, the two, i.e., MIAHDFPGIGHK and HGYPKDIYSS, the relatively short ones, most promising for MMP-1 determination, were selected, mainly considering their advantageous outermost location in the protein molecule and stability against aggregation. MIPs templated with selected epitopes of the MMP-1 protein were successfully prepared by potentiodynamic electropolymerization and simultaneously deposited as thin films on electrodes. The chemosensors, constructed of MIP films integrated with EG-FET, proved useful in determining these epitopes even in a medium as complex as a control serum. The limit of detection for the MIAHDFPGIGHK and HGYPKDIYSS epitope was ∼60 and 20 nM, respectively. Moreover, the chemosensors selectively recognized whole MMP-1 protein in the 50-500 nM concentration range in buffered control serum samples.

Oligonucleotide Analogs and Mimics for Sensing Macromolecular Biocompounds.

Living organisms create life-sustaining macromolecular biocompounds including biopolymers. Artificial polymers can selectively recognize biocompounds and are more resistant to harsh physical, chemical, and physiological conditions than biopolymers are. Due to recognition at a molecular level, molecularly imprinted polymers (MIPs) provide powerful tools to correlate structure with biological functionality and are often used to build next-generation chemosensors. We envision an increasing emergence of nucleic acid analogs (NAAs) or biorelevant monomers built into nature-mimicking polymers. For example, if nucleobases bearing monomers arranged by a complementary template are polymerized to form NAAs, the resulting MIPs will open up novel perspectives for synthesizing NAAs. Despite their usefulness, it is still challenging to use MIPs to devise adaptive biomaterials and to implement them in point-of-care testing.

Promoting bioanalytical concepts in genetics: A TATA box molecularly imprinted polymer as a small isolated fragment of the DNA damage repairing system.

We demonstrate that a new, stable, artificial TATA (T - thymine, A - adenine) box is recognized by amino acids recognizing the natural TATA box. Here, the former mimicked, as a minimal motif, oligodeoxyribonucleotide interactions with amino acids of proteins involved in repairing of damaged dsDNA. By electropolymerization, we molecularly imprinted non-labeled 5'-TATAAA-3' via Watson-Crick nucleobase pairing, thus synthesizing, in a one-step procedure, the hexakis[bis(2,2'-bithien-5-yl)] TTTATA and simultaneously hybridizing it with the 5'-TATAAA-3' template. That is, a stable dsDNA analog having a controlled sequence of nucleobases was formed in the molecularly imprinted polymer (MIP). The 5'-TATAAA-3' was by the X-ray photoelectron spectroscopy (XPS) depth profiling found to be homogeneously distributed both in the bulk of the MIP film and on its surface. The 5'-TATAAA-3' concentration in the 2.8(±0.2)-nm relative surface area, ~140-nm thick MIP film was 2.1 mM. The MIP served as a matrix of an artificial TATA box with the TATAAA-promoter sequence. We comprehensively characterized this artificial DNA hybrid by the polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) and X-ray photoelectron spectroscopy (XPS). Further, we examined interactions of DNA repairing TATA binding protein (TBP) amino acids with the artificial TATA box prepared. That is, molecules of l-phenylalanine aromatic amino acid were presumably engaged in stacking interactions with nucleobase steps of this artificial TATA box. The nitrogen-to­phosphorus atomic % ratio on the surface of the MIP-(5'-TATAAA-3') film increased by ~1.6 times after film immersing in the l-glutamic acid solution, as determined using the XPS depth profiling. Furthermore, l-lysine and l-serine preferentially interacted with the phosphate moiety of 5'-TATAAA-3'. We monitored amino acids interactions with the artificial TATA box using real-time piezoelectric microgravimetry at a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy under flow injection analysis (FIA) conditions.

Oligonucleotide Determination via Peptide Nucleic Acid Macromolecular Imprinting in an Electropolymerized CG-Rich Artificial Oligomer Analogue.

We devised and fabricated a chemosensor for determination of the genetically relevant 5'-GCGGCGGC-3' (G = guanine; C = cytosine) oligonucleotide. For that, we simultaneously electrosynthesized and electrode-immobilized a sequence-defined octakis(2,2'-bithien-5-yl) DNA hybridizing probe using both a "macromolecular imprinting in polymer strategy" and a sequence-programmable peptide nucleic acid (PNA) template. With electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) transductions under stagnant-solution and flow injection analysis (FIA) conditions, respectively, we determined the above oligonucleotide with 200-pM EIS limit of detection. With its EIS-determined apparent imprinting factor of ∼4.0, the chemosensor was discriminative to both mismatched oligonucleotides and Dulbecco's modified Eagle's medium sample interferences.

Programmed Transfer of Sequence Information into a Molecularly Imprinted Polymer for Hexakis(2,2'-bithien-5-yl) DNA Analogue Formation toward Single-Nucleotide-Polymorphism Detection.

A new strategy of simple, inexpensive, rapid, and label-free single-nucleotide-polymorphism (SNP) detection using robust chemosensors with piezomicrogravimetric, surface plasmon resonance, or capacitive impedimetry (CI) signal transduction is reported. Using these chemosensors, selective detection of a genetically relevant oligonucleotide under FIA conditions within 2 min is accomplished. An invulnerable-to-nonspecific interaction molecularly imprinted polymer (MIP) with electrochemically synthesized probes of hexameric 2,2'-bithien-5-yl DNA analogues discriminating single purine-nucleobase mismatch at room temperature was used. With density functional theory modeling, the synthetic procedures developed, and isothermal titration calorimetry quantification, adenine (A)- or thymine (T)-substituted 2,2'-bithien-5-yl functional monomers capable of Watson-Crick nucleobase pairing with the TATAAA oligodeoxyribonucleotide template or its peptide nucleic acid (PNA) analogue were designed. Characterized by spectroscopic techniques, molecular cavities exposed the ordered nucleobases on the 2,2'-bithien-5-yl polymeric backbone of the TTTATA hexamer probe designed to hybridize the complementary TATAAA template. In that way, an artificial TATAAA-promoter sequence was formed in the MIP. The purine nucleobases of this sequence are known to be recognized by RNA polymerase to initiate the transcription in eukaryotes. The hexamer strongly hybridized TATAAA with the complex stability constant KsTTTATA-TATAAA = ka/kd ≈ 106 M-1, as high as that characteristic for longer-chain DNA-PNA hybrids. The CI chemosensor revealed a 5 nM limit of detection, quite appreciable as for the hexadeoxyribonucleotide. Molecular imprinting increased the chemosensor sensitivity to the TATAAA analyte by over 4 times compared to that of the nonimprinted polymer. The herein-devised detection platform enabled the generation of a library of hexamer probes for typing the majority of SNP probes as well as studying a molecular mechanism of the complex transcription machinery, physics of single polymer molecules, and stable genetic nanomaterials.

Inherently Chiral Spider-Like Oligothiophenes.

The racemate of an inherently chiral "spider-like" octathiophene monomer T83 , in which chirality is generated by torsion in its backbone, was synthesized. The racemate was resolved into configurationally stable antipodes by HPLC on a chiral stationary phase. Electrooxidation of the enantiomers resulted in materials displaying high enantiorecognition ability towards the antipodes of some chiral probes. Moreover, the T83 racemate demonstrated great aptitude to stimulate formation of 3D rigid architectures if used as a cross-linking monomer for molecular imprinting. This feature was exploited to devise a molecularly imprinted polymer-based chemosensor selective for a thymine-adenine oligonucleotide.

Molecularly imprinted polymers for separating and sensing of macromolecular compounds and microorganisms.

The present review article focuses on gathering, summarizing, and critically evaluating the results of the last decade on separating and sensing macromolecular compounds and microorganisms with the use of molecularly imprinted polymer (MIP) synthetic receptors. Macromolecules play an important role in biology and are termed that way to contrast them from micromolecules. The former are large and complex molecules with relatively high molecular weights. The article mainly considers chemical sensing of deoxyribonucleic acids (DNAs), proteins and protein fragments as well as sugars and oligosaccharides. Moreover, it briefly discusses fabrication of chemosensors for determination of bacteria and viruses that can ultimately be considered as extremely large macromolecules.