Immobilization Techniques for Aptamers on Gold Electrodes for the Electrochemical Detection of Proteins: A Review.

The development of reliable biosensing platforms plays a key role in the detection of proteins in clinically and environmentally derived samples for diagnostics, as well as for process monitoring in biotechnological productions. For this purpose, the biosensor has to be stable and reproducible, and highly sensitive to detect potentially extremely low concentrations and prevent the nonspecific binding of interfering compounds. In this review, we present an overview of recently published (2017-2019) immobilization techniques for aptamers on gold electrodes for the electrochemical detection of proteins. These include the direct immobilization of thiolated aptamers and the utilization of short linkers, streptavidin/biotin interaction, as well as DNA nanostructures and reduced graphene oxide as immobilization platforms. Applied strategies for signal amplification and the prevention of biofouling are additionally discussed, as they play a crucial role in the design of biosensors. While a wide variety of amplification strategies are already available, future investigations should aim to establish suitable antifouling strategies that are compatible with electrochemical measurements. The focus of our review lies on the detailed discussion of the underlying principles and the presentation of utilized chemical protocols in order to provide the reader with promising ideas and profound knowledge of the subject, as well as an update on recent discoveries and achievements.

An aptamer-based biosensor for detection of doxorubicin by electrochemical impedance spectroscopy.

An aptamer-based biosensor was developed for the detection of doxorubicin using electrochemical impedance spectroscopy. Doxorubicin and its 14-dehydroxylated version daunorubicin are anthracyclines often used in cancer treatment. Due to their mutagenic and cardiotoxic effects, detection in groundwater is desirable. We developed a biosensor using the daunorubicin-binding aptamer as biological recognition element. The aptamer was successfully co-immobilized with mercaptohexanol on gold and a density of 1.3*1013 ± 2.4*1012 aptamer molecules per cm2 was achieved. The binding of doxorubicin to the immobilized aptamer was detected by electrochemical impedance spectroscopy. The principle is based on the inhibition of electron transfer between electrode and ferro-/ferricyanide in solution caused by the binding of doxorubicin to the immobilized aptamer. A linear relationship between the charge transfer resistance (R ct ) and the doxorubicin concentration was obtained over the range of 31 nM to 125 nM doxorubicin, with an apparent binding constant of 64 nM and a detection limit of 28 nM. With the advantages of high sensitivity, selectivity, and simple sensor construction, this method shows a high potential of impedimetric aptasensors in environmental monitoring. Graphical abstract Measurement chamber and immobilization principle for the detection of doxorubicin by electrochemical impedance spectroscopy.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus.

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 1013 aptamer molecules per cm². As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL-1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23µM. The apparent dissociation constant was determined as 13.98µM and the LoD is 3.17µM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD.

Front end with offset-free symmetrical current source optimized for time domain impedance spectroscopy.

Fast impedance measurements are often performed in time domain utilizing broad bandwidth excitation signals. Other than in frequency domain measurements harmonic distortion cannot be compensated which requires careful design of the analog front end. In order to minimize the influence of electrode polarization and noise, especially in low-frequency measurements, current injection shows several advantages compared to voltage application. Here, we show an active front end based on a voltage-controlled current source for a wide range of impedances. Using proper feedback, the majority of the parasitic capacitances are compensated. The bandwidth ranges from dc to 20 MHz for impedance magnitude below 5 kΩ. The output is a symmetric signal without dc-offset which is accomplished by combination of a current conveyor and a voltage inverter. An independent feedback loop compensates the offset arising from asymmetries within the circuitry. We focused especially on the stability of the current source for usage with small metal electrodes in aqueous solutions. At the monitor side two identical, high input impedance difference amplifiers convert the net current through the object and the voltage dropping across into a 50 Ω symmetric output. The entire circuitry is optimized for step response making it suitable for fast time domain measurements.

On the association of interparental conflict with developing behavioral inhibition and behavior problems in early childhood.

Associations between interparental conflict and the development of temperament characteristics have seldom been addressed; moreover, studies on the impact of interparental conflict on developing behavior problems in infancy and early childhood are relatively rare. The authors therefore aimed to contribute to the study of these issues. A sample of 64 families was investigated longitudinally. At the infant age of 4 months, negative emotionality was measured with a laboratory routine. When infants were 4, 8, and 12 months old, maternal sensitivity was assessed during home observations, and interparental conflict was measured by means of questionnaires. When infants were 30 months old, behavioral inhibition was measured in 2 laboratory fear episodes, and behavior problems were assessed with a structured clinical interview of the primary caregiver. Interparental conflict and infant negative emotionality were significantly and independently associated with subsequent behavioral inhibition. The association of interparental conflict with behavior problems was moderated by infant negative emotionality. Infants high in negative emotionality developed more serious behavior problems when exposed to high interparental conflict, whereas the association was not significant in infants low in negative emotionality.

Negative emotionality, attachment quality, and behavior problems in early childhood.

Negative emotionality, as well as attachment security and disorganization, are seen as major contributors to social adjustment and maladjustment in childhood. However, relatively little is known about whether infant negative emotionality and attachment quality operate together to affect developing behavior problems. The present study thus aims to contribute to this question. Participants were 64 healthy firstborn children and their primary caregivers. Negative emotionality was assessed at the infant ages of 4, 8, and 12 months using laboratory routines. At 18 months, the Strange Situation procedure was conducted to assess infant attachment security and disorganization, and at 30 months, the child's behavior problems were assessed within a structured clinical interview. Attachment security and attachment disorganization were significantly associated with subsequent behavior problems. There was no significant relation between infant negative emotionality and behavior problems. However, there were indications of a stronger association between attachment disorganization and behavior problems in infants high in negative emotionality. The results underpin the importance of attachment quality as well as negative emotionality in social adjustment. Disorganized attachment precedes poor adjustment, especially in infants high in negative emotionality.

New type of dry substances content meter using microwaves for application in biogas plants.

Dry substances (DS) are an important index for monitoring and controlling anaerobic co-digestion in biogas plants. We have developed and tested an online meter that measures suspended solids by means of the reflection coefficient of an exiting microwave signal, which is dependent on the dielectric properties of the suspensions. Intelligent models based on partial least squares regression (PLSR) and artificial neural network (ANN) for calibration allow exact and reproducible measurements under different circumstances. This measuring method is appropriate for contactless and online measurements of dry substance contents in biogas plants in a large range from 2-14%.

[Comparing assessment methods of infant emotionality]. / Ein Vergleich unterschiedlicher Erfassungsmethoden der frühkindlichen Emotionalität.

OBJECTIVES: The concept of infant emotionality (temperament) is frequently used in the fields of developmental psychopathology, developmental psychology, and child and adolescent psychiatry. However, a valid assessment of the construct has to deal with some difficulties. Parent reports and behaviour observations of the interaction between caregiver and infant may be biased by parental characteristics, while laboratory assessment procedures often have not been sufficiently validated. METHODS: In the present study, three dimensions of temperament were assessed at three ages during the first year of life using three different measurement approaches. Convergent validity, discriminant validity and the associations of the temperament measures with maternal characteristics, i.e. depression, anxiety and educational status were analyzed. The study group consisted of 101 healthy first-born infants and their primary caregivers. At the ages of 4, 8 and 12 months, positive emotionality, negative emotionality and withdrawal/anxiety were assessed by means of a parent questionnaire, naturalistic behavioural observations of the caregiver-infant interaction, and by means of laboratory routines. RESULTS: Aside from just two exceptions, there were significant convergent correlations between the different measures at each age. Particularly the laboratory routines revealed a good degree of discriminant validity. Questionnaire scores often correlated with the mothers' characteristics. These correlations were independent of the associations with the observational data. CONCLUSIONS: Therewith, parent reports include an objective as well as a subjective component.

Predicting the development of infant emotionality from maternal characteristics.

Few studies have examined the associations between environmental conditions and developing infant emotionality or the differential susceptibility to those conditions. The present longitudinal study aims to make a contribution to close that gap. We analyzed whether positive emotionality, negative emotionality/irritability, and withdrawal/fear at the end of the first year of life are predictable from preceding caregiver's depression/anxiety, social support, and sensitivity in the interaction with the infant while controlling for antecedent states of emotionality. Furthermore, the question of whether associations between maternal characteristics and subsequent fear are stronger in the subgroup of infants high in irritability as opposed to those who are low in irritability was investigated. Subjects were 101 healthy firstborn infants and their primary caregivers. Assessments were conducted at infant ages of 4, 8, and 12 months. Depression, anxiety, and the social support of the caregiver were assessed by questionnaire. Sensitivity in the caregiver-infant interaction was assessed by behavior observations within the scope of home visits. Temperament characteristics were observed in standardized laboratory episodes. Whereas negative emotionality and withdrawal/fear were significantly predictable from the maternal characteristics, no predictability could be shown for developing positive emotionality. There were indications of a stronger association between the maternal characteristics and developing withdrawal/fear in irritable infants.