M. Monsur Ali

Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.

Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection

Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed ‘Integrated Comprehensive Droplet Digital Detection’ (IC 3D) that can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.

Colorimetric Sensing by Using Allosteric-DNAzyme-Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe†

Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

Detection of DNA using bioactive paper strips.

Paper strips containing DNA-conjugated microgels (MG) are used to achieve sensitive DNA detection in three steps: target DNA promoted ligation of a DNA primer to the MG-bound DNA, rolling circle amplification (RCA) between the primer and a circle DNA, and hybridization of the RCA products and a fluorescent DNA probe.

Novel Molecular and Nanosensors for In Vivo Sensing

In vivo sensors are an emerging field with the potential to revolutionize our understanding of basic biology and our treatment of disease. In this review, we highlight recent advances in the fields of in vivo electrochemical, optical, and magnetic resonance biosensors with a focus on recent developments that have been validated in rodent models or human subjects. In addition, we discuss major challenges in the development and translation of in vivo biosensors and present potential solutions to these problems. The field of nanotechnology, in particular, has recently been instrumental in driving the field of in vivo sensors forward. We conclude with a discussion of emerging paradigms and techniques for the development of future biosensors.

DNA-Scaffolded Multivalent Ligands to Modulate Cell Function

We report a simple, versatile, multivalent ligand system that is capable of specifically and efficiently modulating cell‐surface receptor clustering and function. The multivalent ligand is made of a polymeric DNA scaffold decorated with biorecognition ligands (i.e., antibodies) to interrogate and modulate cell receptor signaling and function. Using CD20 clustering‐mediated apoptosis in B‐cell cancer cells as a model system, we demonstrated that our multivalent ligand is significantly more effective at inducing apoptosis of target cancer cells than its monovalent counterpart. This multivalent DNA material approach represents a new chemical biology tool to interrogate cell receptor signaling and functions and to potentially manipulate such functions for the development of therapeutics.

A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection

Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA.

Cell-surface sensors: lighting the cellular environment

Cell-surface sensors are powerful tools to elucidate cell functions including cell signaling, metabolism, and cell-to-cell communication. These sensors not only facilitate our understanding in basic biology but also advance the development of effective therapeutics and diagnostics. While genetically encoded fluorescent protein/peptide sensors have been most popular, emerging cell surface sensor systems including polymer-, nanoparticle-, and nucleic acid aptamer-based sensors have largely expanded our toolkits to interrogate complex cellular signaling and micro- or nano-environments. In particular, cell-surface sensors that interrogate in vivo cellular microenvironments represent an emerging trend in the development of next generation tools which biologists may routinely apply to elucidate cell biology in vivo and to develop new therapeutics and diagnostics. This review focuses on the most recent development in areas of cell-surface sensors. We will first discuss some recently reported genetically encoded sensors that were used for monitoring cellular metabolites, proteins, and neurotransmitters. We will then focus on the emerging cell surface sensor systems with emphasis on the use of DNA aptamer sensors for probing cell signaling and cell-to-cell communication.

Modulation of DNA-Modified Gold-Nanoparticle Stability in Salt with Concatemeric Single-Stranded DNAs for Colorimetric Bioassay Development

Gold nanoparticles (AuNPs) exhibit distinct optical properties under specific conditions. For example, spherical AuNPs of 13 nm in diameter appear red when well dispersed and blue or purple when aggregated. Two major methods used to control DNA-modified AuNP aggregation are DNA-templated assembly (also known as cross-linking aggregation) and salt-induced aggregation. The first approach modulates AuNP stability through sequence-specific hybridization and has been widely explored for biosensing applications. The second approach modulates AuNP stability by using divalent metal ions that neutralize the negative charges of DNA on AuNP surfaces, and has also been exploited to develop various colorimetric bioassays. DNA-conjugated AuNPs have also been used to make organized nanostructures with long single-stranded DNA molecules (ssDNAs) produced by rolling circle amplification (RCA). RCA is an isothermal process of amplifying DNA by using a short DNA primer, a circular ssDNA template (DNA circle) and Phi29 DNA polymerase. Because ssDNA products from RCA contain many repeating (concatemeric) units, RCA technique has been widely used to achieve sensitive detection of DNA or other biological targets. In a recent study, AuNPs and RCA have been employed together for the development of a colorimetric assay to detect single-nucleotide polymorphisms through a crosslinking strategy. Herein, we report on the novel use of RCA products to protect AuNPs against salt-induced aggregation. AuNPs modified with a short ssDNA complementary to part of an RCA product (RP) can organize onto the RP through sequence-specific hybridization. We have found that, when MgCl2 (salt) is added, the AuNP–RP assemblies form unique, globular, island-like nanostructures (Figure 1 a, lower path), which we term “NP islands (NPI)”. The color of the NPI-containing solution remains red whereas in the absence of RP, AuNPs form purple aggregates (Figure 1 a, upper path). We have also exploited this finding for the development of a colorimetric assay for ATP detection wherein the action of an ATP-dependant DNAzyme (aptazyme) is coupled to RCA and the resulted RP is used to protect AuNPs from salt-induced aggregation. AuNPs used in this study (NP1, Figure 1 b) were functionalized with a short ssDNA to a surface density of approximately 50 strands per particle. Optimization of MgCl2 concentration revealed that evident red-to-purple color transition of NP1 solution occurred at 30 mm of MgCl2 (along with 20 mm Tris-HCl, pH 7.5 at 23 8C, 100 mm NaCl); thus, this salt condition was used throughout our study. Two RCA products, RP1 (containing NP1-binding site; blue letters in Figure 1 b) and RP2 (which does not bind NP1), were prepared by using the relevant circular DNA templates and primers (for their sequences, see Figure S1a in the Supporting Information) and analyzed by agarose gel electrophoresis (Figure S1 b). Complex formation between RP1 and NP1 (but not between RP2 and NP1) was also confirmed by agarose gel electrophoresis (Figure S1 c). Before addition of MgCl2, the NP1-only, NP1–RP2, and NP1–RP1 solutions (samples 1–3, respectively) all appeared red (tube inserts in Figure 2 a, 1–3). TEM analysis revealed that gold nanoparticles in samples 1 and 2 were well dispersed (Figure 2 a, 1 and 2) whereas those in sample 3 were clustered (Figure 2 a, 3), suggesting that NP1s in this sample assembled onto RP1 by sequence-specific hybridization. With the addition of 30 mm MgCl2, samples 1 and 2 turned purple (tube inserts in Figure 2 a, 4 and 5) whereas sample 3 remained red (tube insert in Figure 2 a, 6). TEM analysis produced interesting results: huge NP aggregates were found in samples 1 and 2 (Figure 2 a, 4 and 5), howev[a] Dr. M. M. Ali, P. Kanda, S. D. Aguirre, Prof. Dr. Y. Li Department of Biochemistry and Biomedical Sciences and Department of Chemistry and Chemical Biology McMaster University, 1200 Main Street West, Hamilton (Canada) Fax: (+1) 905-522-9033 E-mail : liying@mcmaster.ca Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201002677.

Enzymatic manipulations of DNA oligonucleotides on microgel: towards development of DNA–microgel bioassays

We demonstrate that DNA oligonucleotides covalently coupled to colloidal microgel can be manipulated by T4 DNA ligase for DNA ligation and by Phi29 DNA polymerase for rolling circle amplification (RCA). We also show that the long single-stranded RCA product can generate intensive fluorescence upon hybridization with complementary fluorescent DNA probe. We believe DNA-microgel conjugates can be explored for the development of DNA based bioassays and biosensors.