Formation Of Stacks Research Articles

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

A Review of Conductive Carbon Materials for 3D Printing: Materials, Technologies, Properties, and Applications.

Carbon material is widely used and has good electrical and thermal conductivity. It is often used as a filler to endow insulating polymer with electrical and thermal conductivity. Three-dimensional printing technology is an advance in modeling and manufacturing technology. From the forming principle, it offers a new production principle of layered manufacturing and layer by layer stacking formation, which fundamentally simplifies the production process and makes large-scale personalized production possible. Conductive carbon materials combined with 3D printing technology have a variety of potential applications, such as multi-shape sensors, wearable devices, supercapacitors, and so on. In this review, carbon black, carbon nanotubes, carbon fiber, graphene, and other common conductive carbon materials are briefly introduced. The working principle, advantages and disadvantages of common 3D printing technology are reviewed. The research situation of 3D printable conductive carbon materials in recent years is further summarized, and the performance characteristics and application prospects of these conductive carbon materials are also discussed. Finally, the potential applications of 3D printable conductive carbon materials are concluded, and the future development direction of 3D printable conductive carbon materials has also been prospected.

NO2-Affinitive Conjugated Polymer for Selective Sub-Parts-Per-Billion NO2 Detection in a Field-Effect Transistor Sensor.

Conjugated polymers (CPs) have provided versatile semiconducting implements for the development of soft electronic devices. When three CPs with the same conjugated framework but different side chains were adopted in the field-effect transistor (FET) sensor for NO2 detection, the response to NO2 showed an opposite tendency to the charge carrier mobility of each CP. Morphological and structural characterizations revealed that the flexible glycol side chain enhances NO2 affinity as well as prevents the formation of lamellar stacking of the CP chains, thereby providing routes for the facile diffusion of NO2. Additionally, theoretical calculations for CP-NO2 complex formation at the molecular level support the relatively low energy barrier for inter-chain transition of NO2 between the glycol-based conjugated frameworks, which implies the spontaneous internal diffusion of NO2 to the semiconductor-dielectric interface in the FET-based sensor. As a result, the CP with a NO2-affinitive morphology exhibited an exceptional sensitivity of 13.8%/ppb upon NO2 (100 ppb) exposure for 50 s and provided excellent selectivity to the FET-based sensor toward other environmentally abundant harmful gases, such as SO2, CO2, and NH3. In particular, the theoretic limit of detection reached down to 0.24 ppb, which is the lowest value ever reported for organic FET-based NO2 gas sensors.

Adjusting compressive properties of open-cell Mg-Gd-Zn foams by variation of Gd content

Open-cell Mg-xGd-2Zn (x = 4, 6 and 8 wt.%) foams were fabricated. The phase compositions, microstructures and element distributions of the foams with different amounts of added Gd were studied by X-ray diffraction, optical microscopy, scanning electron microscopy, transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The compressive properties of the foams were evaluated by compressive testing at the temperatures ranging from room temperature (RT) to 300 °C. The experimental results show that an increase in the Gd content refines the microstructures of the foams and promotes the formation of long periodic stacking ordered phase. An increased temperature results in the reduction in the compressive strength and the energy absorption capacity of the foams. Additionally, the compressive strength and energy absorption capacity at each testing temperature increase with increasing Gd content. The mechanisms underlying these effects are discussed based on the relationship between the compressive properties and the Gd content of the foams.

(Invited) Interface Treatment Related Defects During Ge Gate Stack Formation

Germanium as a high mobility channel material is currently of considerable interest since Ge has both higher electron and hole mobility as compared to silicon (1). The deposition of high-k dielectrics on Ge has been a significant challenge because of high interface defects density, Dit (2). By considering the interface reaction kinetics, thermal budget various atomic layer deposition (ALD) schemes and interface treatments can be identified to get a stable dielectric with a robust interface. However, understanding of the interface kinetics and defect distribution into gate stack and/or Ge is still limited.In this work, we report the impact of different interface treatments on the p type Ge prior to any high-k layer deposition on defect generation. Ge surface was oxidized by a controlled chemical oxidation, slot-plane-antenna plasma oxidation and oxidation through vapor O3 treatment before ALD deposition of 1nm Al2O3/3.5nm ZrO2 for gate stack formation. These devices were characterized to evaluate the interface and possible bulk defects by deep level transient spectroscopy (DLTS), capacitance spectroscopy, conductance method in addition to standard I-V and C-V measurements. DLTS has the advantage to have the spectrum comparison among the interface treatments. Signature of these spectra can reveal the difference, which is more accurate than the defects estimated by Conductance method. DLTS also provides the signature of slow traps (~1ms) (Fig. 1) originating from the bulk or interface depending on the type of GeOx formation.Furthermore, we evaluate the defect distribution when the gate stack was subjected to a slot-plane antenna plasma oxidation prior to metal deposition. A TiN/Hf1-xZrxO2/Al2O3/Ge gate stack with six different Zr content was investigated (Fig. 2). Al2O3 was used to impede the Ge diffusion into the gate stack. Interface state density (Dit) and C-V hysteresis show that the mid-gap Dit tend to increase with decrease in EOT, possibly due to the undesirable native oxide formation on Ge.The author would like to thank Drs. Y.M. Ding (Western Digital Corporation, Shanghai, China), M.N. Bhuyian (Globalfoundries, Malta, NY, USA), K.L. Ganapathi (Indian Institute of Technology Madras, India), N. Bhat, (Indian Institute of Science, Bangalore, India) and K. Tapily, R. D. Clark, S. Consiglio, C. S. Wajda, G. Nakamura, and G. J. Leusink of TEL Technology Center, America, Albany, NY, for their help in this work. References Arimura et al, IEDM Technical Digest, (2020)N. Bhuyian et al, ECS Journal of Solid-St. Sci. and Tech., vol. 7(2), N1-N6, (2018)G. Kolla et al, J. Vac. Sci. Technol. B, 36(2), 021201 (2018)Ding et al, IEEE Trans. on Dev. and Materials Reliability, 17(2), 349-354, (2017)Ding, et al, J. of Vac. Sci. and Technol. B, 34(2), 021203, 2016. Figure 1

Stacking of 2D materials containing a thin layer of hexagonal boron nitride using polycaprolactone

To date, even though many transfer methods for 2D materials have been developed, direct transfer of very thin (< 5 nm) hexagonal boron nitride (hBN) and a monolayer of hBN has been elusive. Herein, a novel transfer method for hBN, including monolayer hBN, is introduced that can form various stacks with many 2D materials. Polycaprolactone (PCL) was selected for the method due to its low melting point and softness at room temperature. With PCL, the stacking method was easily controlled, allowing the formation of many stacks with graphene and hBN. When 2D material transfer was attempted using PCL of various molecular weights, higher molecular weight PCL yielded better results. With increasing average molecular weight, the tensile strength, elongation, and adhesion strength of the PCL sample increased. Higher molecular weight and lower molecular weight distribution assisted in the easy and clean transfer of 2D materials.

(Invited) Interface Treatment Related Defects During Ge Gate Stack Formation

The impact of different interface treatments on defect generation during gate stack formation on the p-type germanium is reported in this paper. In most cases 1nm Al2O3 was used to limit the germanium diffusion into the high-k layer. Defects were characterized by conductance method and deep level transient spectroscopy. A signature of slow traps (~1ms) originating from the bulk or interface depending on the type of GeOx formation was observed. Furthermore, the defect formation due to different types interfacial layer formation is also investigated when the gate stacks were subjected to slot plane antenna plasma oxidation. It was observed that a stable GeOx formation leads to a reduction in defect formation at the interface.

Oxidation degree dependent adsorption of ssDNA onto graphene-based surface* *Project supported by the National Natural Science Foundation of China (Grant Nos. 11305237 and 11974366), the Fundamental Research Funds for the Central Universities, China, the Natural Science Foundation of Shanghai, China (Grant No. 19ZR1463200), and the Key Research Program of Chinese Academy of Sciences (Grant No. QYZDJ-SSW-SLH053).

DNA/GO composite plays a significant role in the research field of biotechnology and nanotechnology, and attracts a great deal of interest. However, it is still unclear how the oxidation degree of the graphene-based surface affects the adsorption process of single-strand DNA (ssDNA). In this paper, based on the molecular dynamics simulations, we find that ssDNA molecule is absorbed on the GO surface in the most stable state with the oxidation degree around 15%. The microscopic mechanism is attributed to the van Der Walls and the electrostatic interactions between the ssDNA molecule and the graphene-based surface, which is accompanied with the π–π stacking and hydrogen bond formation. The number of π–π stacking between ssDNA and GO reaches the maximum value when the oxidation degree is around 15% among all the GO surfaces. Our simulation results also reveal the coexistence of stretched and curved configurations as well as the adsorption orientation of ssDNA on the GO surface. Furthermore, it is found that the absorbed ssDNA molecules are more likely to move on the graphene-based surface of low oxidation degree, especially on pristine graphene. Our work provides the physics picture of ssDNA’s physisorption dynamics onto graphene-based surface and it is helpful in designing DNA/GO nanomaterials.

DNA-Mediated Stack Formation of Nanodiscs.

Membrane-scaffolding proteins (MSPs) derived from apolipoprotein A-1 have become a versatile tool in generating nano-sized discoidal membrane mimetics (nanodiscs) for membrane protein research. Recent efforts have aimed at exploiting their controlled lipid protein ratio and size distribution to arrange membrane proteins in regular supramolecular structures for diffraction studies. Thereby, direct membrane protein crystallization, which has remained the limiting factor in structure determination of membrane proteins, would be circumvented. We describe here the formation of multimers of membrane-scaffolding protein MSP1D1-bounded nanodiscs using the thiol reactivity of engineered cysteines. The mutated positions N42 and K163 in MSP1D1 were chosen to support chemical modification as evidenced by fluorescent labeling with pyrene. Minimal interference with the nanodisc formation and structure was demonstrated by circular dichroism spectroscopy, differential light scattering and size exclusion chromatography. The direct disulphide bond formation of nanodiscs formed by the MSP1D1_N42C variant led to dimers and trimers with low yield. In contrast, transmission electron microscopy revealed that the attachment of oligonucleotides to the engineered cysteines of MSP1D1 allowed the growth of submicron-sized tracts of stacked nanodiscs through the hybridization of nanodisc populations carrying complementary strands and a flexible spacer.

Technology Stack Selection Model for Software Design of Digital Platforms

The article is dedicated to the development of a mathematical model and methodology for evaluating the effectiveness of integrating information technology solutions into digital platforms using virtual simulation infrastructures. The task of selecting a stack of technologies is formulated as the task of selecting elements from sets of possible solutions. This allows us to develop a mathematically unified approach to evaluating the effectiveness of different solutions, such as choosing programming languages, choosing Database Management System (DBMS), choosing operating systems and data technologies, and choosing the frameworks used. Introduced technology compatibility operation and decomposition of the evaluation of the efficiency of the technology stack at the stages of the life cycle of the digital platform development allowed us to reduce the computational complexity of the formation of the technology stack. A methodology based on performance assessments for experimental research in a virtual software-configurable simulation environment has been proposed. The developed solution allows the evaluation of the performance of the digital platform before its final implementation, while reducing the cost of conducting an experiment to assess the characteristics of the digital platform. It is proposed to compare the characteristics of digital platform efficiency based on the use of fuzzy logic, providing the software developer with an intuitive tool to support decision-making on the inclusion of the solution in the technology stack.

Polarization influences the evolution of nucleobase-graphene interactions.

In recent years, graphene has attracted attention from researchers as an atomistically thin solid state material for the study on the self-assembly of nucleobases. Non-covalent interactions between nucleobases and graphene sheets play a fundamental role in understanding the self-assembly of nucleobases on the graphene sheet. A fundamental understanding of the effect of molecular polarizability on these non-covalent interactions between the nucleobases and the underlying graphene sheet is absent in the literature. In this paper, we present the results from polarizable molecular dynamics simulation studies to understand the effect of polarization on the strength of non-covalent interactions. To this end, we report the development of Drude parameters for describing the polarizable graphene sheet. The developed parameters were used to study the self-aggregation phenomenon of nucleobases on a graphene support. We observe a significant change in the interaction patterns upon the inclusion of polarization into the system, with polarizable simulations yielding results that closely resemble the experimental studies. Two of the key observations were the probability of the formation of stacks in guanine-rich systems, and the spontaneous formation of H-bonded structures over the graphene sheet, which allude to the importance of the DNA sequence and composition. Both these effects were not observed in the additive simulations. The present study sheds light on the effect of polarization on the adsorption of DNA nucleobases on a graphene sheet, but the methodology can be extended to include a variety of small molecules and complete DNA strands.

Self-Assembled Multi- and Single-Chain Glyconanoparticles and Their Lectin Recognition.

In this work, we describe the physicochemical characterization of amphiphilic glycopolymers synthesized via copper(0)-mediated reversible-deactivation radical polymerization (Cu-RDRP). Depending on the chemical composition of the polymer, these glycopolymers are able to form multi-chain or single-chain polymeric nanoparticles. The folding of these polymers is first of all driven by the amphiphilicity of the glycopolymers and furthermore by the supramolecular formation of helical supramolecular stacks of benzene-1,3,5-tricarboxamides (BTAs) stabilized by threefold hydrogen bonding. The obtained polymeric nanoparticles were subsequently evaluated for their lectin-binding affinity toward a series of mannose- and galactose-binding lectins via surface plasmon resonance. We found that addition of 2-ethylhexyl acrylate to the polymer composition results in compact particles, which translates to a reduction in binding affinity, whereas with the addition of BTAs, the relation between the nature of the particle and the binding ability system becomes more unpredictable.

High surface area Nanoflakes of P-gC3N4 photocatalyst loaded with Ag nanoparticle with intraplanar and interplanar charge separation for environmental remediation

The photocatalytic performance of gC3N4 is majorly restricted by insufficient collection of photogenerated charges on the surface during reaction due to highly dense stacking of lamellar structures with lateral size ranging in microns. This deficiency can be overcome by forming thin nanoflakes by systematically breaking the weak bonds that hold the gC3N4 framework without destroying the basic heptazine unit. With this aim, herein, a combination of three different strategies was implemented to design and develop, Ag-loaded and P-doped gC3N4 nanoflakes (Ag3-P1-NF-gC3N4). Using a systematic synthesis method, bulk gC3N4 was first converted into thin nanosheets, followed by fragmentation into nanoflakes, with a planar size up to 100 nm. P doping to replace the corner C atoms in the gC3N4 matrix (forming PN bonds) and intercalation of plasmonic Ag nanoparticles within the interlayers also assists in the bifurcation of the stacked layers and formation of nanoflake morphology. These strategies result in a significant increase in BET surface area to ∼196 m2/g from 12 m2/g of bulk gC3N4. Improved inter-planar and intra-planar charge mobility was recorded as a result of the reduced sizes. Doping with P also causes higher absorption of the visible spectrum in gC3N4 while the formation of heterojunction with Ag nanoparticles induces efficient separation of photo-generated charges. All these promoting photo-physical properties lead to an outstanding photocatalytic activity towards degradation of aqueous pollutants with reaction rates ∼20 times higher than bulk gC3N4. Complete mineralization of the pollutant and formation of non-toxic byproducts was also confirmed with suitable chromatography techniques.

Polyphenylene as an Active Support for Ru-Catalyzed Hydrogenolysis of 5-Hydroxymethylfurfural.

Selective transformation of biomass feedstocks to platform molecules is a key pursuit for sustainable chemical production. Compared to petrochemical processes, biomass transformation requires the defunctionalization of highly polar molecules at relatively low temperatures. As a result, catalysts based on functional organic polymers may play a prominent role. Targeting the hydrogenolysis of the platform chemical 5-hydroxymethylfurfural (5-HMF), here, we design a polyphenylene (PPhen) framework with purely sp2-hybridized carbons that can isolate 5-HMF via π-π stacking, preventing hemiacetal and humin formation. With good swellability, the PPhen framework here has successfully supported and dispersed seven types of metal particles via a newly developed swelling-impregnation method, including Ru, Pt, Au, Fe, Co, Ni, and Cu. Ru/PPhen is studied for 5-HMF hydrogenolysis, achieving a 92% yield of 2,5-dimethylfuran (DMF) under mild conditions, outperforming the state-of-the-art catalysts reported in the literature. In addition, PPhen helps perform a solventless reaction, achieving direct 5-HMF to DMF conversion in the absence of any liquid solvent or reagent. This approach in designing support-reactant/solvent/metal interactions will play an important role in surface catalysis.

Predicting the efficiency of polyethylene glycol-functionalised graphene in delivery of temozolomide anticancer drug and investigating the effect of pH on the drug release process: DFT and free energy calculations

ABSTRACT The adsorption mechanism of temozolomide (TMZ) anticancer drug on functionalised graphene nanosheet with polyethylene glycol (PEG-GNS) is investigated using density functional theory (DFT) calculations and molecular dynamics (MD) simulations. The probability of the formation of stacking, hydrogen bond, and the simultaneous effect of these two interactions are investigated. The obtained results from the DFT calculations showed that interaction of TMZ with PEG-GNS is exothermic, and stacking interaction between the aromatic rings of TMZ and GNS surface has a vital role in the adsorption process. Also, it is found that the most stable complex is formed in a system where the drug can interact with the graphene surface and PEG agent via stacking and hydrogen bond interactions. Furthermore, MD simulations are performed to understand the effects of pH variation on the adsorption process. The obtained results illustrated that the interaction of the drug with the proposed carrier in the neutral system is stronger than protonated systems. The binding free energy value is reduced by decreasing the pH level. It is observed that interactions between TMZ and the nanocarrier are strong inbiological pH conditions. While these interactions become weaker in the acid condition.

All 2D Heterostructure Tunnel Field-Effect Transistors: Impact of Band Alignment and Heterointerface Quality.

Van der Waals heterostructures are the ideal material platform for tunnel field-effect transistors (TFETs) because a band-to-band tunneling (BTBT) dominant current is feasible at room temperature (RT) because of ideal, dangling bond-free heterointerfaces. However, achieving subthreshold swing (SS) values lower than 60 mV dec-1 of the Boltzmann limit is still challenging. In this work, we systematically studied the band alignment and heterointerface quality in n-MoS2 channel heterostructure TFETs. By selecting a p+-MoS2 source with a sufficiently high doping level, stable gate modulation to a type III band alignment was achieved regardless of the number of MoS2 channel layers. For the gate stack formation, it was found that the deposition of Al2O3 as the top gate introduces defect states for the generation current under reverse bias, while the integration of a hexagonal boron nitride (h-BN) top gate provides a defect-free, clean interface, resulting in the BTBT dominant current even at RT. All 2D heterostructure TFETs produced by combining the type III n-MoS2/p+-MoS2 heterostructure with the h-BN top-gate insulator resulted in low SS values at RT.

Functional analyses of STIM1 mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome.

Tubular aggregate myopathy (TAM) is a progressive disorder characterized by muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), combining TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and STRMK arise from gain-of-function mutations in STIM1 (stromal interaction molecule 1) or ORAI1, both encoding key regulators of Ca2+ homeostasis, and mutations in either gene result in excessive extracellular Ca2+ entry. The pathomechanistic similarities and differences between TAM and STRMK are only partially understood. Here we provide functional in vitro experiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, increase cytosolic Ca2+ levels, promote major nuclear import of the Ca2+ -dependent transcription factor NFAT (nuclear factor of activated T cells), and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+ entry, highlighting that TAM and STRMK involve a common pathomechanism.

Analytical modelling of tantalum/titanium oxide‐based multi‐layer selector to eliminate sneak path current in RRAM arrays

One selector-one resistor (1S-1R) configuration is desirable to use in conductive bridge resistive random-access memory (CBRAM) and resistive random-access memory (RRAM) crossbar arrays (CBAs) to reduce sneak path current. In this study, an analytical model of Ta2O5/TaOx/TiO2 selector device is developed and is integrated with RRAM model to demonstrate the acquired features of 1S-1R to reduce the sneak path current. The proposed selector model is developed by considering the electric field-driven tunnelling mechanisms co-exist in thin multi-layer devices such as direct and Fowler-Nordheim tunnelling. The simulated characteristics of proposed model shows high non-linearity (∼1600), high selectivity (∼104), high current density (∼107 A/cm2) and low off current (∼46 nA). Further, the proposed model is simulated with different top electrode metals and dielectric materials to demonstrate the formation of optimal stack for the desired application. Then, the proposed selector model (1S) is integrated with RRAM model (1R) and the compatibility of the devices is verified. Moreover, from the presented 1S-1R model, various parameters for the establishment of CBA such as read/write voltages for selected/unselected trails are predicted and substantial conditions for sneak path current reduction such as non-linearity, Roff/Ron ratio and off-current (10 nA) are also evaluated.

Non-Covalent π-Stacking Interactions between Asphaltene and Porphyrin in Bitumen.

In recent years, the dominant organizing role of non-covalent π-stacking interactions in the association of asphaltenes and porphyrins was criticized and replaced with cooperative forces that are mostly covalent in nature. Here, we show the significant contribution of non-covalent forces in stabilizing the π-stacking of asphaltenes and porphyrins. To understand the binding chemistry of metalloporphyrin-asphaltene, the interaction of nickel octaethylporphyrin with a series of model fragments for asphaltene was studied in two different pathways: axial coordination and π-stacking. Nickel octaethylporphyrin was specifically studied because a main fraction of vanadium and nickel metals in petroleum residues are chelated with porphyrins, and the refining processes in petroleum industries are affected by the significant detrimental impact of these metal compounds. The results of the extended transition state-natural orbital of chemical valence (ETS-NOCV) technique provide strong evidence that the bonding interaction in the π-stacking configuration is much preferred to the axial coordination. Energy decomposition analysis verifies the significant contribution of non-covalent forces in stabilizing the π-stacking of asphaltene-porphyrin, showing that there are other forces driving the formation of asphaltene-porphyrin stacks. Indeed, a non-negligible portion of these stabilizing forces is contributed by strong orbital mixing interactions through charge transfer between active centers; this contribution is mostly overlooked in π-stacking interactions. This matter includes the π-stacking interactions of asphaltene-asphaltene. Isosurfaces of deformation density (Δρ) provide better insights into the π-stacking preference. NOCV deformation densities are delocalized over the entire complex in the π-stacking conformer, leading to the multi-centric charge transfer zone; Δρ isosurfaces of axial coordination are mostly localized on the limited centers involved in chemical bonding.

An Enhanced Segmentation Technique for Smokers RBC Rouleax Coin Stacking

Smoking is one of the major factors of having coin stacking formation of Red Blood Cell or commonly known as RBC that can cause blood clots and can lead to stroke. Smokers tends have thicker, high count, and overlapping of RBC compared to non-smokers. Blood cell detection plays significant part in biomedical field. There are two methods in detection of RBC, manual inspection by medical experts and automated machines. The manual inspection process is detection of blood cells under a microscope that is more prone to human error and time consuming, while automated hardware solutions like automated haematology machines are available, due to high cost it is not widely available to poor and developing countries who have a high statistic of smokers. Smokers have a high tendency of overlapping RBC or commonly known as rouleaux coin stacking cell formation. This study presents an enhanced segmentation technique that can detect the high degree of overlapping RBCs of smokers using digital image processing that can be helpful in the medical field.