Quantum dots are time bomb: Multiscale toxicological study.

The use of quantum dots has spread widely into many applications. Works on the study of quantum dots on living organisms have had conflicting results on toxicity. There are no full-scale long-term toxicological studies with multiple administration of quantum dots. Understanding the toxicity of quantum dots is still limited. Here we present data on the effects of quantum dots on animals. In this work for the first time, it is shown that at a single administration of quantum dots in the body they have moderate species-specific toxicity, but repeated administration of quantum dots for 14 days even in the amount of 0.5 mg/kg leads to a delayed not completely irreversible hematotoxic effect, delayed irreversible disorders of barrier function of the liver, irreversible nephrotoxic effect, and to pathological changes in the thymus, kidneys and spleen. Administration of quantum dots in the amount of 2.5 mg/kg for 14 days leads to irreversible changes in the lungs, liver, spleen, kidneys and thyroid gland. This phenomenon is based on immunological reactions. On the one hand, these data confirm that quantum dots at a single administration can show relatively low toxicity. On the other hand, they cause to a delayed irreversible organ and tissue damage when repeatedly administered to the body even in small quantities. This study demonstrates that quantum dots are not as low in toxicity as previously thought to be and pose a serious risk when entering living organisms. Detecting and treating poisoning using standard methods of diagnosis and treatment of heavy metal poisoning may not be effective. This study demonstrates that toxic effects of quantum dots on a living body are quite complex and cannot be generalized based on previously reported assumptions.

Use of folic acid nanosensors with excellent photostability for hybrid imaging.

Sentinel lymph node (SLN) mapping and tumor-boundary delineation play a key role in cancer surgery, as they have great potential to reduce surgical intervention and increase relapse-free survival rates of patients. The autofluorescence imaging (AFI) method can improve the efficiency of tumor delineation and optimize the scope of surgical intervention, but there are still no fluorescent drugs that can be used with such a method to form a hybrid imaging technique. Another problem is bleaching when fluorescent dyes are conjugated with folic acid. This study reports, for the first time, nanosensors with excellent photostability and compatibility with endoscopes for AFI, which makes simultaneous hybrid imaging possible. After functionalization of the quantum dot (QD) surfaces, we found that they bound effectively to MCF-7 cancer cells. The diagnostic value of simultaneous hybrid imaging using common AFI equipment in delineating tumor boundaries and mapping SLN can reduce the cost of diagnosis and increase its reliability.

A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus.

A direct double antibody lateral flow assay (DDA-gB-LFA) for the detection of antibodies against the glycoprotein B (gB) of Aujeszky's disease virus (ADV) in swine sera was developed. A native ADV gB was used for the preparation of a conjugate with colloidal gold particles and the immobilization on the strip membrane. The gB purified from ADV virions by immunoaffinity chromatography retained its native epitope structure after adsorption on the nitrocellulose membrane and the surface of colloidal gold particles. The diagnostic specificity and sensitivity of the DDA-gB-LFA were evaluated using 236 field swine sera. The diagnostic specificity and sensitivity of the DDA-gB-LFA compared to a commercially available gB-based ELISA were 98.0% and 98.6%, respectively, when determined with the use of the reader-detection mode, and 98.0% and 93.5%, respectively, when determined using visual detection. The DDA-gB-LFA provides a rapid, sensitive, and specific determination of ADV gB-directed antibodies in sera and can be used for the detection of ADV-exposed swine.

Biodistribution of intact fluorescent CdSe/CdS/ZnS quantum dots coated by mercaptopropionic acid after intravenous injection into mice.

Semiconductor quantum dots (QD) have been widely used for fluorescent bioimaging. However their biosafety has attracted increasing attention, since the data about their in vivo behavior in biological systems are still limited. In this paper we have investigated the short- and long-term biodistribution of intact fluorescent CdSe/CdS/ZnS QD coated by 3-mercaptopropionic acid in mice. The results showed that intravenously injected QD accumulated mainly in the lungs, liver and spleen and were retained in these tissues for over 22 days. QD caused signs of acute toxicity in mice including death. The investigated QD possibly caused vascular thrombosis. The results of a toxicological assay indicated that some histopathological changes occurred in the lung tissue after the injection of QD. Our study highlights the need for careful evaluation of QD safety before their use in biological applications.

FRET-based biosensor for oleic acid in nanomolar range with quantum dots as an energy donor.

A self-assembling sensor for oleic acid has been developed. The sensor consists of a self-assembling fluorescent dye labeled BSA and quantum dots CdSe/ZnS capped with 3-mercaptopropionic acid. The detection limit of the new sensor is 10-1000 nM. The influence of the quantum dot size on the FRET efficiency in the course of the interaction of the sensor system with the analyte has been studied. The pH dependence, aggregation stability. and electrophoretic properties of the sensor have been examined. The data suggest a new approach for the development of nanoscale FRET-based sensors operating effectively due to unique fluorescent properties of quantum dots as well as due to selective protein-ligand interactions.

Comparison of functional properties of mammalian DNA polymerase lambda and DNA polymerase beta in reactions of DNA synthesis related to DNA repair.

DNA polymerase lambda (Pol lambda) is a novel enzyme of the family X of DNA polymerases. Pol lambda has some properties in common with DNA polymerase beta (Pol beta). The substrate properties of Pol lambda were compared to Pol beta using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol lambda was investigated. Pol lambda is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol lambda. FEN1 processes nicked DNA, thus removing a barrier to Pol lambda DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol lambda. Photocrosslinking and functional assay show that Pol lambda is less efficient than Pol beta in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol lambda though it stimulates strand displacement synthesis catalyzed with Pol beta. It is suggested that Pol lambda plays a role in the SP BER rather than contributes to the LP BER pathway.

A new highly efficient photoreactive analogue of dCTP. Synthesis, characterization, and application in photoaffinity modification of DNA binding proteins.

A new base-substituted analogue of dCTP, exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) has been synthesized and characterized. FAP-dCTP is an efficient substrate of mammalian DNA polymerase beta in the reaction of primer elongation displaying substrate properties as an analogue of dCTP and dTTP. FAP-dCTP was used for the photoaffinity modification of mammalian DNA polymerase beta. Two approaches to photoaffinity labeling were utilized. In one approach, photoreactive FAP-dCTP was first incorporated into radiolabeled primer-template, and photoreactive DNA was UV-irradiated in the presence of DNA polymerase beta, which resulted in the polymerase labeling by photoreactive primer. In an alternate approach, FAP-dCTP was first UV-cross-linked to the enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked FAP-dCTP was incorporated into the 3'-end of radioactive primer. This "catalytic" modification pathway was shown to be less specific in recognition of FAP-dCTP as an analogue of dCTP than dTTP. FAP-dCTP was used as substrate of endogenous DNA polymerases of HeLa cell extract to synthesize photoreactive DNAs for photoaffinity modification of cell proteins. UV irradiation results in modification of DNA binding proteins of cell extract. The level of photoaffinity labeling of protein targets in the cell extract was strongly dependent on the efficiency of synthesis of photoreactive DNA.

Analyzing the handoff of DNA from UvrA to UvrB utilizing DNA-protein photoaffinity labeling.

To better define the molecular architecture of nucleotide excision repair intermediates it is necessary to identify the specific domains of UvrA, UvrB, and UvrC that are in close proximity to DNA damage during the repair process. One key step of nucleotide excision repair that is poorly understood is the transfer of damaged DNA from UvrA to UvrB, prior to incision by UvrC. To study this transfer, we have utilized two types of arylazido-modified photoaffinity reagents that probe residues in the Uvr proteins that are closest to either the damaged or non-damaged strands. The damaged strand probes consisted of dNTP analogs linked to a terminal arylazido moiety. These analogs were incorporated into double-stranded DNA using DNA polymerase beta and functioned as both the damage site and the cross-linking reagent. The non-damaged strand probe contained an arylazido moiety coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Six site-directed mutants of Bacillus caldotenax UvrB located in different domains within the protein (Y96A, E99A, R123A, R183E, F249A, and D510A), and two domain deletions (Delta2 and Deltabeta-hairpin), were assayed. Data gleaned from these mutants suggest that the handoff of damaged DNA from UvrA to UvrB proceeds in a three-step process: 1) UvrA and UvrB bind to the damaged site, with UvrA in direct contact; 2) a transfer reaction with UvrB contacting mostly the non-damaged DNA strand; 3) lesion engagement by the damage recognition pocket of UvrB with concomitant release of UvrA.