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L-lactate is a key metabolite indicative of physiological states, glycolysis pathways, and various diseases such as sepsis, heart attack, lactate acidosis, and cancer. Detection of lactate has been relying on a few enzymes that need additional oxidants. In this work, DNA aptamers for L-lactate were obtained using a library-immobilization selection method and the highest affinity aptamer reached a K d of 0.43 mM as determined using isothermal titration calorimetry. The aptamers showed up to 50-fold selectivity for L-lactate over D-lactate and had little responses to other closely related analogs such as pyruvate or 3-hydroxybutyrate. A fluorescent biosensor based on the strand displacement method showed a limit of detection of 0.55 mM L-lactate, and the sensor worked in 90 % serum. Simultaneous detection of L-lactate and D-glucose in the same solution was achieved. This work has broadened the scope of aptamers to simple metabolites and provided a useful probe for continuous and multiplexed monitoring., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Computer-aided prediction of aptamer sequences has been focused on primary sequence alignment and motif comparison. We observed that many aptamers have a conserved hairpin, yet the sequence of the hairpin can be highly variable. Taking such secondary structure information into consideration, a new algorithm combining conserved primary sequences and secondary structures is developed, which combines three scores based on sequence abundance, stability, and structure, respectively. This algorithm was used in the prediction of aptamers from the caffeine and theophylline selections. In the late rounds of the selections, when the libraries were converged, the predicted sequences matched well with the most abundant sequences. When the libraries were far from convergence and the sequences were deemed challenging for traditional analysis methods, this algorithm still predicted aptamer sequences that were experimentally verified by isothermal titration calorimetry. This algorithm paves a new way to look for patterns in aptamer selection libraries and mimics the sequence evolution process. It will help shorten the aptamer selection time and promote the biosensor and chemical biology applications of aptamers.

Since the report of the RNA aptamer for theophylline, theophylline has become a key molecule in chemical biology for designing RNA switches and riboswitches. In addition, theophylline is an important drug for treating airway diseases including asthma. The classic RNA aptamer with excellent selectivity for theophylline has been used to design biosensors, although DNA aptamers are more desirable for stability and cost considerations. In this work, we selected DNA aptamers for theophylline, and all the top sequences shared the same binding motifs. Binding was confirmed using isothermal titration calorimetry and a nuclease digestion assay, showing a dissociation constant ( K d ) around 0.5 μM theophylline. The Theo2201 aptamer can be truncated down to 23-mer while still has a K d of 9.8 μM. The selectivity for theophylline over caffeine is around 250,000-fold based on a strand-displacement assay, which was more than 20-fold higher compared to the classic RNA aptamer. For other tested analogs, the DNA aptamer also showed better selectivity. Using the structure-switching aptamer sensor design method, a detection limit of 17 nM theophylline was achieved in the selection buffer, and a detection limit of 31 nM was obtained in 10% serum.

Tetracyclines are a group of important antibiotics with a common four-ring scaffold. While most tetracyclines are currently used only in animals, their leaching into the environment and residues in food have caused health concerns. Aptamers are an attractive way to detect tetracyclines, and all previously reported aptamers for tetracyclines were obtained by immobilizing target molecules. In this work, we selected a few DNA aptamers by immobilizing the DNA library using oxytetracycline as the target. We obtained new aptamers with no overlapping sequences compared to the previously reported ones, and a representative sequence named OTC5 had a dissociation constant of 147 nM measured by isothermal titration calorimetry. Similar binding affinities were also observed with tetracycline and doxycycline. Because tetracyclines are fluorescent and their fluorescence intensity was enhanced by binding to the aptamers, a label-free and dye-free fluorescent biosensor was developed with a detection limit of 25 nM oxytetracycline. The sensor was able to detect targets in milk after extraction. Fluorescence polarization measurement showed that this aptamer is insensitive to sodium concentration but requires magnesium. Finally, a strand-displacement biosensor was designed, and it has a detection limit of 1.2 μM oxytetracycline.

Ethanolamine is an important analyte for environmental chemistry and biological sciences. A few DNA aptamers were previously reported for binding ethanolamine with a dissociation constant ( K d ) as low as 9.6 nM. However, most of the previous binding assays and sensing work used either immobilized ethanolamine or immobilized aptamers. In this work, we studied three previously reported DNA sequences, two of which were supposed to bind ethanolamine while the other could not bind. Isothermal titration calorimetry revealed no binding for any of these sequences. In addition, due to their guanine-rich sequences, thioflavin T was used as a probe. Little fluorescence change was observed with up to 1 μM ethanolamine. Responses within the millimolar range of ethanolamine were attributed to the general fluorescence quenching effect of ethanolamine instead of aptamer binding. Finally, after studying the adsorption of ethanolamine to gold nanoparticles (AuNPs), we confirmed the feasibility of using AuNPs as a probe when the concentration of ethanolamine was below 0.1 mM. However, no indication of specific aptamer binding was observed by comparing the three DNA sequences for their color changing trends. This work articulates the importance of careful homogeneous binding assays using free target molecules.

Highly sensitive and selective detection of lanthanide ions is a major analytical challenge. In recent years, the use of DNA for this purpose has been pursued. For such highly charged cations, it is difficult to select their aptamers due to strong nonspecific binding. On the other hand, the use of catalytic DNA or DNAzymes has an advantage to overcome this problem, especially DNAzymes with RNA-cleaving activity. In this chapter, a few such DNAzymes are introduced and methods for in vitro selection of lanthanide-dependent RNA-cleaving DNAzymes are described in detail, including the selection protocols, the DNA sequences used, the characterization of selected DNAzymes and their conversion into biosensors. All of the experiments use only fluorophore-labeled DNA, and radioisotope labeling is completely avoided. The resulting DNAzymes can distinguish lanthanides from non-lanthanide metals, tell the difference between light and heavy lanthanides, and can be used together to discriminate individual lanthanides., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nickel is a highly important metal, and the detection of Ni 2+ using biosensors is a long-stand analytical challenge. DNA has been widely used for metal detection, although no DNA-based sensors were reported for Ni 2+ . DNAzymes are DNA-based catalysts, and they recruit metal ions for catalysis. In this work, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 50 random nucleotides in the presence of Ni 2+ . To increase Ni 2+ binding, a glycyl-histidine-functionalized tertiary amine moiety was inserted at the cleavage junction. A representative DNAzyme named Ni03 showed a high cleavage yield with Ni 2+ and it was further studied. After truncation, the optimal sequence of Ni03l could bind one Ni 2+ or two Co 2+ for catalysis, while other metal ions were inactive. Its cleavage rates for 100 μM Ni 2+ reached 0.63 h -1  at pH 8.0. A catalytic beacon biosensor was designed by labeling a fluorophore and a quencher on the Ni03l DNAzyme. Fluorescence enhancement was observed in the presence of Ni 2+ with a detection limit of 12.9 μM. The sensor was also tested in spiked Lake Ontario water achieving a similar sensitivity. This is another example of using single-site modified DNAzyme for sensing transition metal ions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

GR5 is the first reported DNAzyme, which has RNA cleavage activity in the presence of Pb2+. Due to its excellent selectivity, GR5 has been a popular DNAzyme for developing biosensors for Pb2+. The activity of DNAzymes is often affected by pH and salt, which may in turn affect the sensitivity of related sensors. Although pH can be readily controlled by using a buffer, the effect of salt is more complex. To have a systematic understanding, we herein measured the cleavage activity of GR5 in various concentrations of Na+ and Mg2+. Both metals inhibited the DNAzyme with Pb2+, and the inhibition constants were 1.8 mM Mg2+ and 33.4 mM Na+. For anions, F- inhibited GR5 more strongly than Br-, while Cl- was the least inhibiting anion, which was consistent with the solubility of their lead salts. The reaction can work similarly in many Good's buffers, while phosphate buffer should be avoided. Finally, GR5 is an optimal sequence based on the truncation and elongation studies. This study reveals important solution conditions that should be considered when designing and testing related biosensors for metal detection.

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ca 2+ is a highly important metal ion in biology and in the environment, and thus there is extensive work in developing sensors for Ca 2+ detection. Although many Ca 2+ -binding proteins are known, few nucleic acids can selectively bind Ca 2+ . DNA-based biosensors are attractive for their high stability and excellent programmability. We report a RNA-cleaving DNAzyme, EtNa, cooperatively binding two Ca 2+ ions but to only one Mg 2+ . Four DNAzymes with known Ca 2+ -dependent activity were compared, and the EtNa had the best selectivity for Ca 2+ . The EtNa is 90 times more active in Ca 2+ than in Mg 2+ . Phosphorothioate (PS) modification showed that both non-bridging oxygen atoms at the scissile phosphate contribute equally to Ca 2+ binding. The pH-rate profile suggests two concurrent deprotonation reactions. EtNa was further engineered for Ca 2+ sensing, and found to have a detection limit of 17 μm Ca 2+ and excellent selectivity. The detection of Ca 2+ in tap water was performed, and the result was comparable with that by ICP-MS. This study offers new fundamental insights into Ca 2+ binding by nucleic acids and improved metal selectivity by having multiple cooperative metal binding sites., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mercury is a highly toxic heavy metal and many DNA-based biosensors have been recently developed for Hg(2+) detection in water. Among them, thymine-rich DNA is the most commonly used for designing Hg(2+) sensors. However, the thymine-Hg(2+) interaction is strongly affected by the buffer conditions. We recently reported a molecular beacon containing phosphorothioate (PS)-modified RNA linkages that can be cleaved by Hg(2+). In this work, the fluorescence quenching and DNA adsorption properties of nano-sized graphene oxide (NGO) were used to develop a new sensor using the PS-RNA chemistry. Three DNA probes, containing one, three and five PS-RNA linkages, respectively, were tested. Finally, a fluorophore-labeled poly-A DNA with five PS-RNA linkages was selected and adsorbed by NGO. In the presence of Hg(2+), the fluorophore was released from NGO due to the cleavage reaction, resulting in a fluorescence enhancement. This sensor is highly selective for Hg(2+) with a detection limit of 8.5 nM Hg(2+). For comparison, a fluorophore-labeled poly-T DNA was also tested, which responded to Hg(2+) more slowly and was inhibited by high NaCl concentrations, while the PS-RNA probe was more tolerant to different buffer conditions. This work indicates a new method for interfacing DNA with NGO for Hg(2+) detection.

Graphene oxide adsorbs single-strand fluorescent probe DNA, and the adsorbed probe can be desorbed by adding the complementary target DNA. Using this method, many biosensor studies have been carried out. We recently proposed a two-step mechanism for this sensing reaction: non-specific probe displacement followed by hybridization in the solution. Only about one out of six added target DNA is hybridized with the adsorbed probe to generate signal, leading to relatively low sensitivity. In this work, we aim to test whether surface blocking agents can minimize non-specific target adsorption and increase hybridization efficiency. Over ten blocking agents (polymers, surfactants, and DNA) were screened based on their effect on probe DNA adsorption and target DNA induced probe desorption. DNA oligonucleotides show significant and controllable enhancement in sensor sensitivity. The effect of DNA length and sequence was systematically investigated. Under optimized conditions, the sensor sensitivity was enhanced by nearly 10-fold. Using the same blocking method, sensitivity enhancement of other targets was also achieved, including adenosine and Hg(2+) with DNA aptamer probes. This reported surface blocking strategy can generally improve graphene oxide and potentially other surface adsorption based biosensors for metal ions, small molecules, and DNA., (Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Graphene oxide (GO) has attracted extensive research interest as a platform for DNA adsorption and biosensor development. While most researchers use simple physisorption of fluorescently labeled DNA, covalent sensors are less susceptible to non-specific probe displacement and minimize false positive results. In this work, three thymine-rich DNA probes of different lengths are modified on their 3'-end with an amino group for covalent conjugation to GO. They also each contain an internally labeled fluorophore so that Hg(2+) binding can lead to a large distance increase between the fluorophore and the GO surface for fluorescence signaling. Hg(2+)-dependent fluorescence signaling from the covalent sensors are compared with that from the non-covalent sensors in terms of sensitivity, selectivity, signaling kinetics, and continuous monitoring. The covalent sensors are much more stable and resistant to non-specific probe displacement, while still retaining high sensitivity and similar selectivity. The detection limits are 16.3 and 20.6 nM Hg(2+), respectively, for the covalent and non-covalent sensors, and detection of spiked Hg(2+) in Lake Ontario water is demonstrated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cu(2+) is a very important metal ion in biology, environmental science, and industry. Developing biosensors for Cu(2+) is a key topic in analytical chemistry. DNAzyme-based sensors are highly attractive for their excellent sensitivity, stability, and programmability. In the past decade, a few Cu(2+) biosensors were reported using DNAzymes with DNA cleavage or DNA ligation activity. However, they require unstable ascorbate or imidazole activation. So far, no RNA-cleaving DNAzymes specific for Cu(2+) are known. In this work, a phosphorothioate (PS) RNA-containing library was used for in vitro selection, and a few new Cu(2+)-specific RNA-cleaving DNAzymes were isolated. Among them, a DNAzyme named PSCu10 was studied further. It has only eight nucleotides in the enzyme loop with a cleavage rate of 0.1 min(-1) in the presence of 1 μM Cu(2+) at pH 6.0 (its optimal pH). Between the two diastereomers of the PS RNA chiral center, the R(p) isomer is 37 times more active than the S(p) one. Among the other divalent metal ions, only Hg(2+) can cleave the substrate due to its extremely high thiophilicity. A catalytic beacon sensor was designed with a detection limit of 1.6 nM Cu(2+) and extremely high selectivity. PSCu10 is specific for Cu(2+), and it has no cleavage in the presence of ascorbate, which reduces Cu(2+) to Cu(+).

RNA-cleaving DNAzymes provide a unique platform for developing biosensors. However, a majority of the work has been performed in clean buffer solutions, while the activity of some important DNAzymes in biological sample matrices is still under debate. Two RNA-cleaving DNAzymes (17E and 10-23) are the most widely used. In this work, we carefully studied a few key aspects of the 17E DNAzyme in human blood serum, including hybridization, cleavage activity, and degradation kinetics. Since direct fluorescence monitoring is difficult due to the opacity of serum, denaturing and nondenaturing gel electrophoresis were combined for studying the interaction between serum proteins and DNAzymes. The 17E DNAzyme retains its activity in 90% human blood serum with a cleavage rate of 0.04 min(-1), which is similar to that in the PBS buffer (0.06 min(-1)) with a similar ionic strength. The activity in serum can be accelerated to 0.3 min(-1) with an additional 10 mM Ca(2+). As compared to 17E, the 10-23 DNAzyme produces negligible cleavage in serum. Degradation of both the substrate and the DNAzyme strand is very slow in serum, especially at room temperature. Degradation occurs mainly at the fluorophore label (linked to DNA via an amide bond) instead of the DNA phosphodiester bonds. Serum proteins can bind more tightly to the 17E DNAzyme complex than to the single-stranded substrate or enzyme. The 17E DNAzyme hybridizes extremely fast in serum. With this understanding, the detection of DNA using the 17E DNAzyme is demonstrated in serum.

Developing biosensors for lanthanides is an important but challenging analytical task. To address this problem, in vitro selection of RNA-cleaving DNAzymes was carried out using a library containing a region of 35 random nucleotides in the presence of Lu(3+), since Lu(3+) was reported to be the most efficient lanthanide for RNA cleavage. The resulting DNA sequences can be aligned to a single family with two conserved stretches of nucleotides. One of the representative DNAzymes (named Lu12) was further studied. Lu12 is more active with smaller lanthanides and has the lowest activity in the presence of the largest lanthanide (lutetium). Its cleavage rate is 0.12 min(-1) in the presence of 10 μM Nd(3+) at pH 6.0. This is a new DNAzyme, and a catalytic beacon sensor is designed by attaching a fluorophore/quencher pair, detecting Nd(3+) down to 0.4 nM (72 parts-per-trillion). This DNAzyme is highly selective for lanthanides as well, showing cleavage only with two nonlanthanide ions: Y(3+) and Pb(2+). We previously reported a DNAzyme named Ce13d, which has similar responses to all the trivalent lanthanides. Combining these two allows for a ratiometric assay that identifies a few large lanthanides.

With the fast growth of cancer research, new analytical methods are needed to measure anticancer drugs. This is usually accomplished by using sophisticated analytical instruments. Biosensors are attractive candidates for measuring anticancer drugs, but currently few biosensors can achieve this goal. In particular, it is challenging to have a general method to monitor various types of anticancer drugs with different structures. In this work, a biosensor was developed to detect anticancer drugs by modifying carbon paste electrodes with glutathione-s-transferase (GST) enzymes. GST is widely studied in the metabolism of xenobiotics and is a major contributing factor in resistance to anticancer drugs. The measurement of anticancer drugs is based on competition between 1-chloro-2,4-dinitrobenzene (CDNB) and the drugs for the GST enzyme in the electrochemical potential at 0.1V vs. Ag/AgCl by square wave voltammetry (SWV) or using a colorimetric method. The sensor shows a detection limit of 8.8μM cisplatin and exhibits relatively long life time in daily measurements., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aptamers are single-stranded nucleic acids that selectively bind to target molecules. Most aptamers are obtained through a combinatorial biology technique called SELEX. Since aptamers can be isolated to bind to almost any molecule of choice, can be readily modified at arbitrary positions and they possess predictable secondary structures, this platform technology shows great promise in biosensor development. Over the past two decades, more than one thousand papers have been published on aptamer-based biosensors. Given this progress, the application of aptamer technology in biomedical diagnosis is still in a quite preliminary stage. Most previous work involves only a few model aptamers to demonstrate the sensing concept with limited biomedical impact. This Critical Review aims to summarize progress that might enable practical applications of aptamers for biological samples. First, general sensing strategies based on the unique properties of aptamers are summarized. Each strategy can be coupled to various signaling methods. Among these, a few detection methods including fluorescence lifetime, flow cytometry, upconverting nanoparticles, nanoflare technology, magnetic resonance imaging, electronic aptamer-based sensors, and lateral flow devices have been discussed in more detail since they are more likely to work in a complex sample matrix. The current limitations of this field include the lack of high quality aptamers for clinically important targets. In addition, the aptamer technology has to be extensively tested in a clinical sample matrix to establish reliability and accuracy. Future directions are also speculated to overcome these challenges.

Nanomaterials for DNA adsorption are useful for sequence-specific DNA detection. Current materials for DNA adsorption employ electrostatic attraction, hydrophobic interaction, or π-π stacking, none of which can achieve sequence specificity. Specificity might be improved by involving hydrogen bonding and metal coordination. In this work, a diverse range of nucleobase/nucleotide (adenine, adenosine, adenosine 5'-triphosphate (ATP), adenosine 5'-monophosphate (AMP), and guanosine 5'-triphosphate (GTP)) coordinated materials containing various metal ions (Au(III), Ag(I), Ce(III), Gd(III), and Tb(III)) are prepared. In most cases, nanoparticles are formed. These materials have different surface charges, and positively charged particles only show nonspecific DNA adsorption. Negatively charged materials give different adsorption kinetics for different DNA sequences, where complementary DNA homopolymers are adsorbed faster than other sequences. Therefore, the bases in the coordinated materials can still form base pairs with the DNA. The adsorption strength is mainly controlled by the metal ions, where Au shows the strongest adsorption while lanthanides are weaker. These materials can be used as sensors for DNA detection and can also deliver DNA into cells with no detectable toxicity. By tuning the nanoparticle formulation, enhanced detection can be achieved. This study is an important step toward rational design of materials to achieve specific interactions between biomolecules and synthetic nanoparticle surfaces.

Many homogeneous assays are complicated by the adsorption of probe molecules by the surface of reaction vessels, which are often made of polypropylene or polystyrene-based plastics. To solve this problem, many protein and surfactant-based blocking agents are used. However, these blockers may interfere with intended assays by sequestering transition-metal ions, inducing protein denaturing, generating air bubbles or making pores in membranes. Coating surfaces with polyethylene glycol (PEG) through covalent linkages has been proven to be an effective method to minimize protein adsorption. However, this method is more difficult to apply on plastic surfaces and is quite expensive. While unmodified PEG is often considered as a nonadsorbing polymer, in this Technical Note, we report that PEG at very low concentration (ppm level) can still effectively block plastic surfaces. This method works for DNA, protein, and liposome-based assays as long as the molecular weight of PEG is greater than 2000. PEG works because of multivalent hydrophobic interaction from its repeating methylene units. This Technical Note will not only facilitate biosensor development, but also enhance our understanding of the interaction between various molecules and plastic surfaces.

Cerium oxide nanoparticles (nanoceria) have recently emerged as a nanozyme with oxidase activity. In this work, we present a few important interfacial properties of nanoceria. First, the surface charge of nanoceria can be controlled not only by adjusting pH but also by adsorption of simple inorganic anions. Adsorption of phosphate and citrate gives negatively charged surface over a broad pH range. Second, nanoceria adsorbs DNA via the DNA phosphate backbone in a sequence-independent manner; DNA adsorption inhibits its oxidase activity. Other anionic polymers display much weaker inhibition effects. Adsorption of simple inorganic phosphate does not have the inhibition effect. Third, nanoceria is a quencher for many fluorophores. These discoveries provide an important understanding for further use of nanoceria in biosensor development, materials science, and nanotechnology.

Silica-encapsulated Raman-reporter embedded SERS nanoprobes, named nanoaggregate embedded beads (NAEBs), were conjugated to the Salmonella specific tailspike protein (TSP) isolated from the P22 bacteriophage to enable a highly specific and ultrasensitive optical transduction platform. We demonstrate three successful surface conjugation strategies and highlight the detection of a single bacterium using SERS., (This journal is © The Royal Society of Chemistry 2012)

A novel hybrid surface-enhanced Raman scattering (SERS) substrate based on Au nanoparticles decorated inverse opal (IO) photonic crystal (PhC) is presented. In addition to the enhancement contributed from Au nanoparticles, a desired Raman signal can be selectively further enhanced by appropriately overlapping the center of photonic bandgap of the IO PhC with the wavelength of the Raman signal. Furthermore, the lattice structure of the IO PhC provides excellent control of the distribution of Au nanoparticles to produce SERS spectra with high uniformity. The new design of SERS substrate provides extra maneuverability for ultra-high sensitivity sensor applications.