Construction of an OCP-ATR-FTIR Spectroscopy Device to In Situ Monitor the Interfacial Reaction of Contaminants: Competitive Adsorption of Cr(VI) and Oxalate on Hematite.

The comprehensive understanding of contaminant interfacial behavior strongly depends on the in situ characterization technique, which is still a great challenge. In this study, we constructed a device integrated with open-circuit potentialand attenuated total reflectance Fourier transform infrared (OCP-ATR-FTIR) spectroscopy to simultaneously monitor the electrochemical and infrared spectral information on the interfacial reaction for the process analysis, taking the competitive adsorption of hexavalent chromium (Cr(VI)) and oxalate on hematite nanocubes (HNC) as an example. The synchronous OCP and infrared results revealed that Cr(VI) interacted with HNC via bidentate binuclear inner-sphere coordination, accompanied by electron transfer from HNC to Cr(VI), while oxalate was adsorbed on HNC through bidentate mononuclear side-on inner-sphere coordination with electron transfer from HNC to oxalate, and also outer-sphere coordination with negative charge accumulation. When oxalate was added to HNC with preadsorbed Cr(VI), oxalate would occupy the inner-sphere adsorption sites and thus cause the detaching of preadsorbed Cr(VI) from HNC. This study provides a promising in situ characterization technique for real-time interfacial reaction monitoring and also sheds light on the competitive adsorption mechanism of oxalate and Cr(VI) on the mineral surface.

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes.

Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azo-enhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 µW/µm2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 102-104 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.

Vibrational Fingerprint Analysis of an Azo-based Resonance Raman Scattering Probe for Imaging Proton Distribution in Cellular Lysosomes.

Due to the fundamental mechanism of vibrational state transitions for chemical bonds, the spectra of Raman scattering are narrow-banded and photostable signals capable of probing specific reactions. In the case of protonation/deprotonation reactions, certain chemical bonds are broken and new bonds are formed. Based on the changes of the vibrational modes for the corresponding bonds, fingerprint analysis of multiple Raman bands may allow for the in situ visualization of proton distribution in live cells. However, Raman scattering faces the well-known challenge of low sensitivity. To perform the vibrational fingerprint analysis of Raman scattering by overcoming this challenge, we developed an azo-based resonance Raman pH probe. It was an azobenzene-featured small molecule responsive to protons with the inherent Raman signal ∼104-fold more intense than that of the conventional alkyne-type Raman reporter 5-ethynyl-2'-deoxyuridine. Through the substitution of the electron-donating and -withdrawing entities to the azobenzene group, the effect of resonance Raman scattering and fluorescence quenching was obtained. This effect resulted in a significant Raman enhancement factor of ∼103 compared to the counterpart molecules without the molecular design. Based on the enhanced Raman sensitivity of the azo-based resonance Raman pH probe, the identification of vibrational fingerprint changes at the azo group was achieved during the protonation/deprotonation reactions, and the vibrational fingerprint analysis resolved a pH difference of less than 0.2 unit. The method enabled sensitive hyperspectral cell imaging that clearly visualized the change of proton distribution in autophagic cells.

Ratiometric Fluorescent Probe for Monitoring Endogenous Methylglyoxal in Living Cells and Diabetic Blood Samples.

Optical imaging provides noninvasive powerful tools not only for better understanding the physiological and pathological roles of methylglyoxal (MGO) in living systems but also for potential clinical diagnosis of MGO-related diseases, such as diabetic complications. However, so far only very few "turn-on" MGO fluorescent sensors have been developed, and they are all based on the reaction between MGO and benzenediamines. Due to the possible reactions of benzenediamines with other cellular molecules, such as NO and FA, these sensors suffer from limited selectivity and potential deactivation in cells. Herein, we report a novel MGO recognition reaction using 2-aminoacetamide. The reaction between MGO and 2-aminoacetamide was found to be highly efficient and specific, with no interference from NO and FA in particular. This reaction was used to develop the first ratiometric fluorescent probe (CMFP) for MGO. We have proven that CMFP could detect MGO at physiological concentrations in both aqueous buffer and living cells with excellent selectivity and sensitivity. Furthermore, we successfully utilized CMFP to study intracellular MGO generation routes and evaluated MGO levels of clinic blood samples from healthy and diabetic patients. These results highlight the potential utility of this probe in both basic science research and clinical diagnosis.

A smart preparation strategy for point-of-care cellular counting of trace volumes of human blood.

Blood counting is one of the most commonly ordered clinical assays, and is often part of the basis for initial diagnosis and screening for disease. While substantial prior research has shown the ability of portable instruments to accurately produce blood counts through image- or flow-based cytometry, these methods require complex sample preparation using either costly commercial imaging chambers or complicated reagents. To address these issues, in this paper we developed a method to prepare trace volumes of whole blood aimed at portable blood counting. The strategy is based on pre-storing dry-form reagents and fabricating a specifically designed cell counter. In order to obtain total cell counts for red blood cells, platelets, and 3-part differentials of white blood cells, two parallel counting chambers with different depths are made from cost- and environmentally friendly materials using soft lithography. As little as 1 µl of whole blood is prepared with pre-stored reagents in centrifuge vials, whereas red blood cells are sphered and white blood cells are stained at the same time. Driven by the capillary force, prepared blood samples enter the hydrophilic chambers automatically. Monolayers of cells are formed when the blood dilution factors and the chamber depths are co-optimized. Combined with our previous custom-built instrument and automated analysis algorithm, the sample preparation strategy allows producing counting results with excellent agreement to a gold-standard clinical hematology instrument. The success of this preparation method may further advance applications of our technology for global use in low-resource settings where central hematology laboratories are not accessible. Graphical abstract Graphical Abstract.

Single-Bead Quantification of Peptide Loading Distribution for One-Bead One-Compound Library Synthesis Using Confocal Raman Spectroscopy.

We report an analytical method to determine peptide loading of "one-bead one-compound" (OBOC) combinatorial peptide libraries at single-bead level. The quantification is based on a linear relationship between the amount of N-terminal amino groups on individual peptide beads and the intensity of Raman signal obtained from a specifically designed reporter labeled on amino groups. Confocal Raman spectroscopy was employed to characterize peptide loading of beads with defined peptide sequences and from OBOC combinatorial peptide libraries. Although amine loading of blank TentaGel beads was found to be uniform, peptide loading among beads of OBOC peptide libraries varied substantially, particularly for those libraries with long sequences. Construction of OBOC libraries can be monitored with this novel analytical technique so that synthetic conditions can be optimized for the preparation of high-quality OBOC peptide libraries. As the variability of peptide loading of individual library beads can significantly influence the screening results, quantitative information obtained by this method will allow us to gain insight into the complexity and challenge of OBOC library synthesis and screening.

Smart and Fast Blood Counting of Trace Volumes of Body Fluids from Various Mammalian Species Using a Compact, Custom-Built Microscope Cytometer.

We report an accurate method to count red blood cells, platelets, and white blood cells, as well as to determine hemoglobin in the blood of humans, horses, dogs, cats, and cows. Red and white blood cell counts can also be performed on human body fluids such as cerebrospinal fluid, synovial fluid, and peritoneal fluid. The approach consists of using a compact, custom-built microscope to record large field-of-view, bright-field, and fluorescence images of samples that are stained with a single dye and using automatic algorithms to count blood cells and detect hemoglobin. The total process takes about 15 min, including 5 min for sample preparation, and 10 min for data collection and analysis. The minimum volume of blood needed for the test is 0.5 µL, which allows for minimally invasive sample collection such as using a finger prick rather than a venous draw. Blood counts were compared to gold-standard automated clinical instruments, with excellent agreement between the two methods as determined by a Bland-Altman analysis. Accuracy of counts on body fluids was consistent with hand counting by a trained clinical lab scientist, where our instrument demonstrated an approximately 100-fold lower limit of detection compared to current automated methods. The combination of a compact, custom-built instrument, simple sample collection and preparation, and automated analysis demonstrates that this approach could benefit global health through use in low-resource settings where central hematology laboratories are not accessible.

Bead-based spontaneous Raman codes for multiplex immunoassay.

In the immunoassay process, for fulfilling the need to identify multiple analytes in a small amount of complex sample matrix, it is desirable to develop highly efficient and specific multiplex suspension array technology. Raman coding strategy offers an attractive solution to code the suspension arrays by simply combing narrow spectral bands with stable signal intensities through solid-phase synthesis on the resin beads. Based on this strategy, we report the bead-based spontaneous Raman codes for multiplex immunoassay. The study resulted in superior selectivity of the Raman-encoded beads for binding with single and multiple analytes, respectively. With the use of mixed types of Raman-encoded immunoassay beads, multiple targets in small amounts of samples were identified rapidly and accurately. By confirming the feasibility of bead-based spontaneous Raman codes for multiplex immunoassay, we anticipate this novel technology to be widely applied in the near future.

Orthogonal Combinatorial Raman Codes Enable Rapid High-Throughput-Out Library Screening of Cell-Targeting Ligands.

High-throughput assays play an important role in the fields of drug discovery, genetic analysis, and clinical diagnostics. Although super-capacity coding strategies may facilitate labeling and detecting large numbers of targets in a single assay, practically, the constructed large-capacity codes have to be decoded with complicated procedures or are lack of survivability under the required reaction conditions. This challenge results in either inaccurate or insufficient decoding outputs. Here, we identified chemical-resistant Raman compounds to build a combinatorial coding system for the high-throughput screening of cell-targeting ligands from a focused 8-mer cyclic peptide library. The accurate in situ decoding results proved the signal, synthetic, and functional orthogonality for this Raman coding strategy. The orthogonal Raman codes allowed for a rapid identification of 63 positive hits at one time, evidencing a high-throughput-out capability in the screening process. We anticipate this orthogonal Raman coding strategy being generalized to enable efficient high-throughput-out screening of more useful ligands for cell targeting and drug discovery.

Raman spectroscopic and microscopic monitoring of on-site and in-situ remediation dynamics in petroleum contaminated soil and groundwater.

The mechanistic study of soil and groundwater remediation in petroleum contaminated lands significantly demands rapid qualitative and quantitative identification of petroleum substances. However, most traditional detection methods cannot provide the on-site or in-situ information of petroleum compositions and contents simultaneously even with multi-spot sampling and complex sample preparation. In this work, we developed a strategy for the on-site detection of petroleum compositions and in-situ monitoring of petroleum contents in soil and groundwater using dual-excitation Raman spectroscopy and microscopy. The detection time was 0.5 h for the Extraction-Raman spectroscopy method and one minute for the Fiber-Raman spectroscopy method. The limit of detection was 94 ppm for the soil samples and 0.46 ppm for the groundwater samples. Meanwhile, the petroleum changes at the soil-groundwater interface were successfully observed by Raman microscopy during the in-situ chemical oxidation remediation processes. The results revealed that hydrogen peroxide oxidation released petroleum from the interior to the surface of soil particles and then to groundwater during the remediation process, while persulfate oxidation only degraded petroleum on the soil surface and in groundwater. This Raman spectroscopic and microscopic method can shed light on the petroleum degradation mechanism in contaminated lands, and facilitate the selection of suitable soil and groundwater remediation plans.

Rapid ultrasensitive detection of hexavalent chromium in soil and groundwater by a microProbing imaging platform.

Rapid detection methods are needed to investigate the environmental quality risk of soil and groundwater in contaminated lands. Currently there is lack of rapid detection methods to sensitively and accurately analyze contaminations of hexavalent chromium in soil due to the challenge of complex sample pretreatment or expensive instrumentation. Here we report a rapid accurate detection platform for quantifying hexavalent chromium in soil and groundwater with ultrasensitivity. The platform consists of a novel sensor of microProbing beads and a portable microscope. Each microProbing bead was a nanoliter reactor to selectively sequestrate Cr (VI) with the enrichment factor up to 150 ×. The microProbing beads presented the signal uniformity of ~97% for the statistical colorimetric imaging analysis. Combined with a miniaturized microscope, the microProbing beads allowed for detecting aqueous Cr (VI) and soluble Cr (VI) in soil within 45 min. The platform achieved high sensitivity with the detection limits of 0.003 ppb for aqueous Cr (VI) and 0.07 ppm for soil Cr (VI). It accurately detected soil and groundwater samples from a chromium contaminated land in Yangtze River Basin of China. The consistency to the laboratory standard methods was achieved with the low cost of ~0.20 US dollar per test. The microProbing imaging platform with the operational simplicity and device portability is highly promising for the field analysis of Cr (VI) in contaminated lands.

Stoichiometry of reconstituted high-density lipoproteins in the hydrated state determined by photon antibunching.

Apolipoprotein A-I plays a central role in the solution structure of high-density lipoproteins. Determining the stoichiometry of lipid-bound apo A-I in the hydrated state is therefore fundamental to understanding how high-density lipoproteins form and function. Here, we use the quantum optical phenomenon of photon antibunching to determine the number of apo A-I molecules bound to discoidal lipoproteins and compare this with values obtained by photon-counting histogram analysis. Both the photon antibunching and photon-counting analyses show that reconstituted high-density lipoprotein particles contain two apo A-I molecules, which is in agreement with the commonly accepted double-belt model.

A new analytical platform for potential point-of-care testing of circulating tumor cells.

It is of significance to detect circulating tumor cells (CTCs) in whole blood using transportable instruments at the point of care to assist evaluating chemotherapeutic efficacy and recurrence risk of cancer patients. However, the current widely used detection methods either require expensive and complex equipments, need complicated enrichment steps, or produce high rates of false positive and/or negative results. Aiming for solving the two critical challenges involved in instrumentation miniaturization and simplification of sample preparation for POCT of CTCs without sacrificing the detection sensitivity and accuracy, this work reports a custom-built, automatic, large field-of-view microscopic CTC cytometer and a novel enrichment strategy based on a synthesized peptide ligand discovered from One-Bead One-Compound library screening. The custom-built microscope has compact size, low weight and efficient cost while still maintaining a detection limit of as low as 5 target objects. The simplified sample preparation utilized a novel peptide LXW7 functionalized to magnetic beads and allows for rapid, highly selective and sensitive detection of CTCs. This analytical platform may fulfill the unmet need for possible point-of-care CTC counting, and provide a new option for early diagnosis of cancers and convenient evaluation of chemotherapeutic efficacy and cancer recurrence.

Azo-Enhanced Raman Scattering for Enhancing the Sensitivity and Tuning the Frequency of Molecular Vibrations.

Raman scattering provides stable narrow-banded signals that potentially allow for multicolor microscopic imaging. The major obstacle for the applications of Raman spectroscopy and microscopy is the small cross section of Raman scattering that results in low sensitivity. Here, we report a new concept of azo-enhanced Raman scattering (AERS) by designing the intrinsic molecular structures using resonance Raman and concomitant fluorescence quenching strategies. Based on the selection of vibrational modes and the enhancing unit of azobenzenes, we obtained a library of AERS molecules with specific Raman signals in the fingerprint and silent frequency regions. The spectral characterization and molecular simulation revealed that the azobenzene unit conjugated to the vibrational modes significantly enhanced Raman signals due to the mechanism of extending the conjugation system, coupling the electronic-vibrational transitions, and improving the symmetry of vibrational modes. The nonradiative decay of azobenzene from the excited state quenched the commitment fluorescence, thus providing a clean background for identifying Raman scattering. The most sensitive AERS molecules produced Raman signals of more than 4 orders of magnitude compared to 5-ethynyl-2'-deoxyuridine (EdU). In addition, a frequency tunability of 10 distinct Raman bands was achieved by selecting different types of vibrational modes. This methodology of AERS allows for designing small-molecule Raman probes to visualize various entities in complex systems by multicolor spontaneous Raman imaging. It will open new prospects to explore innovative applications of AERS in interdisciplinary research fields.

Super-capacity information-carrying systems encoded with spontaneous Raman scattering.

Optical multiplex barcode systems have been significantly boosting the throughput of scientific discovery. A high volume of barcodes can be made from combinations of distinct spectral bands and intensity levels. However, the practical capacity often reaches a ceiling due to the overlaps of signal frequencies or intensities when massive information is written on individual carriers. In this paper, we built super-capacity information-carrying systems by tuning vibrational signals into octal numeral intensities in multiple bands of Raman-silent regions. This novel approach experimentally yielded the largest capacity of distinct optical barcodes to date. The experiments of encoding ASCII and Unicode systems to write and read languages indicate that the Raman coding method provides a new strategy for super-capacity data storage. In addition, multiplex screening of a cell-binding ligand was implemented to demonstrate the feasibility of this technology for fast and in situ high-throughput bio-discovery. These information-carrying systems may open new scenarios for the development of high-throughput screening, diagnostics and data storage.

Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides.

All cells expel a variety of nano-sized extracellular vesicles (EVs), including exosomes, with composition reflecting the cells' biological state. Cancer pathology is dramatically mediated by EV trafficking via key proteins, lipids, metabolites, and microRNAs. Recent proteomics evidence suggests that tumor-associated exosomes exhibit distinct expression of certain membrane proteins, rendering those proteins as attractive targets for diagnostic or therapeutic application. Yet, it is not currently feasible to distinguish circulating EVs in complex biofluids according to their tissue of origin or state of disease. Here we demonstrate peptide binding to tumor-associated EVs via overexpressed membrane protein. We find that SKOV-3 ovarian tumor cells and their released EVs express α3ß1 integrin, which can be targeted by our in-house cyclic nonapeptide, LXY30. After measuring bulk SKOV-3 EV association with LXY30 by flow cytometry, Raman spectral analysis of laser-trapped single exosomes with LXY30-dialkyne conjugate enabled us to differentiate cancer-associated exosomes from non-cancer exosomes. Furthermore, we introduce the foundation for a highly specific detection platform for tumor-EVs in solution with biosensor surface-immobilized LXY30. LXY30 not only exhibits high specificity and affinity to α3ß1 integrin-expressing EVs, but also reduces EV uptake into SKOV-3 parent cells, demonstrating the possibility for therapeutic application.

Expression and Association of the Yersinia pestis Translocon Proteins, YopB and YopD, Are Facilitated by Nanolipoprotein Particles.

Yersinia pestis enters host cells and evades host defenses, in part, through interactions between Yersinia pestis proteins and host membranes. One such interaction is through the type III secretion system, which uses a highly conserved and ordered complex for Yersinia pestis outer membrane effector protein translocation called the injectisome. The portion of the injectisome that interacts directly with host cell membranes is referred to as the translocon. The translocon is believed to form a pore allowing effector molecules to enter host cells. To facilitate mechanistic studies of the translocon, we have developed a cell-free approach for expressing translocon pore proteins as a complex supported in a bilayer membrane mimetic nano-scaffold known as a nanolipoprotein particle (NLP) Initial results show cell-free expression of Yersinia pestis outer membrane proteins YopB and YopD was enhanced in the presence of liposomes. However, these complexes tended to aggregate and precipitate. With the addition of co-expressed (NLP) forming components, the YopB and/or YopD complex was rendered soluble, increasing the yield of protein for biophysical studies. Biophysical methods such as Atomic Force Microscopy and Fluorescence Correlation Spectroscopy were used to confirm that the soluble YopB/D complex was associated with NLPs. An interaction between the YopB/D complex and NLP was validated by immunoprecipitation. The YopB/D translocon complex embedded in a NLP provides a platform for protein interaction studies between pathogen and host proteins. These studies will help elucidate the poorly understood mechanism which enables this pathogen to inject effector proteins into host cells, thus evading host defenses.

Single-step preparation and image-based counting of minute volumes of human blood.

Current flow-based blood counting devices require significant medical infrastructure and are not appropriate for field use. In this article we report on the development of a sample preparation, measurement, and analysis method that permits automated and accurate counting of red blood cells (RBCs), white blood cells (WBCs), and platelets, as well as allowing a 3-part differential of the WBCs to be performed on extremely small volumes of whole blood. This method is compatible with portable instrumentation that can be deployed in the field. The method consists of serially diluting blood samples first with sodium dodecyl sulfate dissolved in phosphate buffered saline, then in acridine orange dissolved in phosphate buffered saline, followed by fluorescence and dark field imaging with low magnification objectives. Image analysis is performed to extract cell counts and differentials. We performed a paired analysis of 20 volunteers with complete blood count values both within and beyond the normal reference range using a commercial automated hematology analyzer and the image-based method, with the new method achieving accuracies comparable to that of the commercial system. Because the sample preparation and imaging are simple and inexpensive to implement, this method has applications for pediatrics, clinician offices, and global health in regions that do not have access to central hematology laboratories.

Tumor-targeting multifunctional micelles for imaging and chemotherapy of advanced bladder cancer.

AIM: This work aimed to determine if the treatment outcomes of bladder cancer could be improved by targeting micelles that are decorated with bladder cancer-specific ligands on the surface and loaded with the chemotherapeutic drug paclitaxel. MATERIALS & METHODS: Targeting efficacy and specificity was determined with cell lines. An in vivo targeting and anti-tumor efficacy study was conducted in mice carrying patient-derived xenografts. RESULTS & DISCUSSION: Targeting micelles were more efficient than nontargeting micelles in delivering the drug load into bladder cancer cells both in vitro and in vivo (p < 0.05). The micelle formulation of paclitaxel was less toxic than free paclitaxel in Cremophor(®) (Sigma, MO, USA) and allowed administration of three-times the maximum tolerated dose without increasing the toxicity. Targeting micelles were more effective than the nontargeting micelles in controlling cancer growth (p = 0.0002) and prolonging overall survival (p = 0.002). CONCLUSION: Targeting micelles loaded with paclitaxel offer strong potential for clinical applications in treating bladder cancer.

Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry.

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps. Telodendrimers (TD) are branched polymers made up of a dendritic oligo-lysine core that is conjugated to linear polyethylene glycol (PEG) on one end, and the lysine "branches" are terminated with cholic acid moieties that enable the formation of nanomicelles in aqueous solution. We report herein that the addition of TD during cell-free synthesis of NLPs produces unique hybrid nanoparticles that have drastically reduced polydispersity as compared to NLPs made in the absence of TD. This finding was supported by dynamic light scattering, fluorescence correlation spectroscopy, and cryo transmission electron microscopy (Cryo-EM). These techniques demonstrate the ability of TDs to modulate both the NLP size (6-30 nm) and polydispersity. The telodendrimer NLPs (TD-NLPs) also showed 80% less aggregation as compared to NLPs alone. Furthermore, the versatility of these novel nanoparticles was shown through direct conjugation of small molecules such as fluorescent dyes directly to the TD as well as the insertion of a functional membrane protein.