biosensors
Article
Electrochemical Response of Saccharomyces cerevisiae
Corresponds to Cell Viability upon Exposure to
Dioclea reflexa Seed Extracts and Antifungal Drugs
Patrick Kobina Arthur 1,2 , Anthony Boadi Yeboah 3, Ibrahim Issah 3 ,
Srinivasan Balapangu 2,3, Samuel K. Kwofie 2,3,4, Bernard O. Asimeng 3, E. Johan Foster 5
and Elvis K. Tiburu 2,3,*
1 Department of Biochemistry, Cell and Molecular Biology, University of Ghana, Legon P.O. Box LG 54,
Ghana; parthur14@gmail.com
2 West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Legon P.O. Box LG 54,
Ghana; srinivasan_bs85@yahoo.com (S.B.); skwofie2000@gmail.com (S.K.K.)
3 Department of Biomedical Engineering, School of Engineering Sciences, College of Basic and Applied
Sciences, University of Ghana, Legon P.O. Box LG 25, Ghana; ayboadi@st.ug.edu.gh (A.B.Y.);
issahi62@gmail.com (I.I.); boasimeng@ug.edu.gh (B.O.A.)
4 Department of Medicine, Loyola University Medical Center, Chicago, IL 60153, USA
5 Department of Materials Science and Engineering, Virginia Tech, Blacksburg, VA 24061, USA; johanf@vt.edu
* Correspondence: etiburu@ug.edu.gh




Received: 10 January 2019; Accepted: 2 March 2019; Published: 20 March 2019 

Abstract: Dioclea reflexa bioactive compounds have been shown to contain antioxidant properties.
The extracts from the same plant are used in traditional medical practices to treat various diseases
with impressive outcomes. In this study, ionic mobility in Saccharomyces cerevisiae cells in the presence
of D. reflexa seed extracts was monitored using electrochemical detection methods to link cell death
to ionic imbalance. Cells treated with ethanol, methanol, and water extracts were studied using cyclic
voltammetry and cell counting to correlate electrochemical behavior and cell viability, respectively.
The results were compared with cells treated with pore-forming Amphotericin b (Amp b), as well
as Fluconazole (Flu) and the antimicrobial drug Rifampicin (Rif). The D. reflexa seed water extract
(SWE) revealed higher anodic peak current with 58% cell death. Seed methanol extract (SME) and
seed ethanol extract (SEE) recorded 31% and 22% cell death, respectively. Among the three control
drugs, Flu revealed the highest cell death of about 64%, whereas Amp b and Rif exhibited cell
deaths of 35% and 16%, respectively, after 8 h of cell growth. It was observed that similar to SWE,
there was an increase in the anodic peak current in the presence of different concentrations of Amp
b, which also correlated with enhanced cell death. It was concluded from this observation that
Amp b and SWE might follow similar mechanisms to inhibit cell growth. Thus, the individual
bioactive compounds from the water extracts of D. reflexa seeds could further be purified and tested
to validate their potential therapeutic application. The strategy to link electrochemical behavior to
biochemical responses could be a simple, fast, and robust screening technique for new drug targets
and to understand the mechanism of action of such drugs against disease models.
Keywords: electrochemical detection; Dioclea reflexa; bioactive; amphotericin; rifampicin; cell viability
1. Introduction
Electrochemical detection of drugs that interact with most biological systems is an important
strategy to understand cellular stresses that cause cell death [1–3]. Saccharomyces cerevisiae (S. cerevisiae)
shares the complex internal cell structure of animal cells and serves as an ideal model for conducting
Biosensors 2019, 9, 45; doi:10.3390/bios9010045 www.mdpi.com/journal/biosensors
Biosensors 2019, 9, 45 2 of 12
research in higher eukaryotes. S. cerevisiae has been used extensively to study cellular mechanisms,
including DNA damage and repair as well as systematic fungal infections [4–6]. Evidence from
previous findings indicate that there are several membrane redox centers in most eukaryotic cells that
can be targeted to monitor redox activities in the presence of certain drugs [7–9].
There are essentially two pathways (lipid-mediated and diffusion porins) through which both
hydrophobic and hydrophilic antimicrobials elicit their potency. The degree of permeation of the cell
membrane has a major impact on the redox activity. In addition, the presence of a hydrophobic drug
within the complex architecture of the membrane induces pore formation and enhances ionic flow,
which can be detected electrochemically. Non-membrane-mediated drugs diffuse freely through the
membrane and may not necessarily destabilize the membrane architecture; therefore, the ionic flow
that can be captured by electrochemical detection techniques is limited.
The construction and maintenance of a high-quality natural products library based on microbial,
plant, marine, or other sources is a resource-intensive endeavor. Crude extract libraries have lower
resource requirements for sample preparation and allow for rapid screening of bioactive compounds.
Until now, the mechanistic studies of natural products through high throughput screening (HTS)
requires high quality natural product library [10,11]. While HTS provide a thorough understanding
of drug behavior that can inform further characterization, the procedures are also resource-intensive.
Screening methods, especially for antimicrobial lead compounds, have been a major challenge, and
a number of modifications to the methods have been made over the years [12]. We therefore intend
to develop a simple procedure based on electrochemistry that allows simple and rapid screening of
membrane-targeted leads. The concept is based on the premises that membrane modulation of a
ligand can offset the chemical balance, thereby enhancing the flow of ions/charges and potentiating
the activities of antimicrobial compounds [6,13]. To test these hypotheses, Dioclea reflexa seed extracts
and three extensively studied antifungal and antibiotic drugs were selected to investigate their
electrochemical behaviors using S. cerevisiae cells as a model biological system.
D. reflexa is a leguminous plant that is commonly found in tropical Africa and South America.
Previous studies on D. reflexa revealed remarkable medicinal properties, including antioxidant and
inflammation activities, which have been exploited to treat a number of diseases with extremely
impressive outcomes [14,15]. The leaves and seeds of the plant have phytochemical compounds,
which possess antimicrobial and antioxidant properties, and are used to treat typhoid, asthma, and
rheumatism [13–15]. However, the detailed mechanism of action of this plant extracts in treating
almost all the diseases mentioned is not fully understood. In this work, ethanol, methanol, and water
extracts of the bioactive molecules in D. reflexa seeds will be used to study the electrochemical behavior
of S. cerevisiae cell lines, and the results are correlated to cell death for identifying potential drug
leads [16].
One of the most extensively used antimicrobial drugs for studying S. cerevisiae is amphotericin b
(Amp b) and fluconazole (Flu), which is used as a fungistatic drug [17–19]. Another drug, rifampicin
(Rif), which is a strong antibiotic against tuberculosis (TB), has also been extensively studied in model
TB strains [20]. Amp b, Flu, and Rif are used as controls. Amp b is a membrane-mediated drug that
increases the permeability of ions and small molecules by binding to ergosterol in the S. cerevisiae
membrane to create pores [21,22]. Flu, on the other hand, has an antifungal influence on Candida
albicans as well as other fungal diseases because it inhibits ergosterol synthesis, whereas Rif binds to
the beta subunit of the DNA-dependent RNA polymerase enzyme complex to inhibit the transcription
of messenger RNA in TB strains. Unlike Amp b and Flu, Rif does not exhibit antifungal activity, but it
can diffuse freely through different organelles. Thus, the electrochemical response of the cells in the
presence of the three extracts will be compared to cells treated with these commercial drugs to validate
the feasibility of such techniques in screening plant products. The aim of this work is to develop ion
channel mimetic biosensors for detecting membrane-targeted natural products using amperometric
response mechanisms.
Biosensors 2019, 9, 45 3 of 12
2. Methods
2.1. Growth Medium and Cell Culturing
A cell culture of S. cerevisiae (ATCC/LGC Standards, Teddington, Middlesex, UK) was maintained
on YEPD agar at 4 ◦C (Yeast extract Peptone, Dextrose, and granulated Agar). Yeast cultures were
grown in 150 mL of YEPD broth in shake flasks rotated at 180 rpm for 16 h at 30 ◦C. The cells were
then harvested by centrifugation at 16,000 rpm and washed twice in 25 mL of 50 mM phosphate
buffer of pH 7. The cells were then re-suspended in sterile phosphate-buffered saline (PBS, 50 mM
K2HPO4/KH2PO4, pH 7, 100 mM KCl) (Sigma-Aldrich, St. Louis, MO, USA). The optical density of
the cell suspension was adjusted to give an OD600 of 40 using an LKB Novaspec 11 spectrophotometer
(Pharmacia Biotech, Piscataway, NJ, USA). The cells were used on the day of harvest at a seeding
density of 2.15 × 103 cells/cm2.
2.2. Seed Drying and Extraction and Drug Acquisition
The fresh seeds of Dioclea reflexa were obtained from a farm in Suma Ahenkro in the District
of Jaman North in the Brong Ahafo region of Ghana (coordinates: 7◦57′1.8” North and 2◦41′52.08”
West). The seeds were identified by Prof. Isaac Kojo Asante, the head of the Department of Botany at
the University of Ghana. The cotyledon inside the pericarp was dried for ten days in the open sun,
after which the seed was cracked open and dried for an additional ten days under room temperature.
When the seed was fully dried, the cotyledon was ground to a powder using a laboratory mortar and
pestle. All commercial drugs were obtained from a vendor (Sigma-Aldrich, Saint Louis, MO, USA).
2.3. Solvent Extraction
Five grams of the seed powder was mixed with 30 mL of each solvent (70% ethanol, 70% methanol,
and 100% deionized water, all solvents were obtained from Sigma-Aldrich, St. Louis, MO, USA with
99.9% purity). The mixture was then rotated on an orbital mixer for 48 h. It was later removed and
then allowed to settle. The supernatants from all the extractions were freeze-dried, and the resulting
powder was reconstituted with 1000 µL of 70% ethanol. UV-VIS absorption measurements were done
using a JENWAY, 6705 UV-Vis Spectrophotometer (Cole-Parmer, Staffordshire, UK).
2.4. Cell Viability Measurements Using Trypan Blue Based Assay
A stock solution of 1 mg/mL of the drug or the extracts were prepared separately using dimethyl
sulfoxide (≥99.7% purity) (Sigma-Aldrich, St. Louis, MO, USA). The final drug or extract concentration
in 200 µL of cells ranges from 5–30 µg/mL [23]. The cells were incubated with the drugs or the extracts
in time intervals ranging from 20 min to 8 h. Twenty microliters of cells were added to 20 µL of 0.2%
trypan blue, prepared in PBS at pH = 7.2 and mixed thoroughly. After which 20 µL of the resulting
solution was pipetted and then deposited onto the counting chamber for the cell viability studies using
a Nexcelom Cellometer (Nexcelom Bioscience, Lawrence, MA, USA).
Electrochemical detection of the cells was done using cyclic voltammetry under steady-state
conditions. A CheapStat potentiostat device (IO Rodeo, Pasadena, CA, USA) was used in all
experiments. Interdigitated Gold Electrodes (IDEs)/Microelectrodes was purchased from Metrohm,
DropSens (Llanero, Spain) and composed of two interdigitated electrodes with two connection tracks
all made of gold on a glass substrate. The design of the Interdigitated electrodes allows two electrodes
to fuse together, and as a result, the distance between two electrodes is reduced. The electrodes were
thoroughly cleaned and polished before each measurement. The potentiostat was held at open circuit
prior to each scan, and the cyclic voltammograms were obtained by scanning from 690 mV to 970 mV
at a scan rate of 10 mV/s. Notably, the position of the voltammogram on the current axis gave an
immediate indication of the proportions of each quantification of the redox form [17].
Biosensors 2019, 9, x FOR PEER REVIEW 4 of 12 
at a scan rate of 10 mV/s. Notably, the position of the voltammogram on the current axis gave an 
immediate indication of the proportions of each quantification of the redox form [17].  
Biosensors 2019, 9, 45 4 of 12
3. Results  
3. Results
3.1. Structure of the Antifungal Drugs and Schematic of the Study 
3.1. StrucTtuhree cohf tehme iAcanlt isfutrnugcatluDrreusg osf aanmd pSchhoetmeraitcicino f(Athme Spt ubd)y, fluconazole (Flu), and rifampicin (Rif) are 
sThhoewcnh ienm Fiicgaulrset r1u. cAtumreps bo fisa am ppohloyteenreic winit(Ah msepvebn) ,afldjuocionninagz otrlean(Fs lduo),uabnled broifnadms.p Ficluin is(R ai fs)yanrtehetic 
showtrniaiznoFlei gwuirteh 1fu. nAgmisptabticis aactpivoiltyye, nwehweriethass Revife ins aa dsejominisiynngtthraetnisc adnotuibbiloetbico onbdtsa.iFnleud ifsroamsy Sntrtehpetotimcyces. 
triazTohlee wstietphwfuisneg pisrtoactiecdaucrteiv init yth, wish weroeraks wRaifsi tsoa psreombeis tyhnet mheetcichaannitsibmio otfi caoctbitoanin oef dthfreo amntSimtriecprtoombiyacle ds.rugs 
The astnedp wpliasnetp erxotcreadcutsre, aisn sthhoiswwno irnk Fwigausrteo 2p. rFoibrestt,h tehem dercuhga/nciosnmstoitfuaecntitosn oof fththee palanntitm eixctrroabcitasl wdreurge sused 
and tpol atnartgeextt rtahcet sm, aesmsbhroawnne einnvFiirgounrem2e.nFti arsntd, t hcaeudsreu mg/ecmonbsrtaitnuee dnetspooflatrhiezaptliaonnt,e lexatrdaicntsg wtoe rteheu fsoerdmtoation 
targeotf tphoermese wmibtrha nane  einncvrieraosnemd epnetramnedacbailuitsye mtoe pmrobtroannse adnedp omlaornizoavtiaolnen, tle iaodnisn sgu
+ +
tcoht haes fNoarm aantido nKo.f The 
poreisonwiict h tarnaninscferre aswedasp ecrampetaubrieldit ytthorporuogtho nselaencdtromcohnemoviaclaeln tdieotnesctsiuocnh afoslNloaw+eadn dbKy+ . cTehlle ivoniaibc ility 
tranmsfeeraswuarsemcaepnttusr teod dthetreorumgihneel tehcetr ocochrreemlaitciaolnd beetetwctieoenn fioolnloicw medobbiylitcye lalcvrioasbsil tithye mbieoalsougriceaml emnetsmtborane 
detearmndin ceeltlh deecaotrhr.e lation between ionic mobility across the biological membrane and cell death.
HO
HO
O
O
OOHH OO
H OOHH OOHH
O
OO NNHH
N
Amphotericin b HOO N N HO
O N O
OH OH N O
Amphotericin B O OOHH OOOH OH N H OOHH OOHH
O O
OO NNHH
N
Rifampicin
N
Amphotericin b  O N N
NAmpNhoterNicin b. N O NOH OH N
HO Amphotericin B O
N OH OH N
O
N
Rifampicin
F F
N N N N
Fluconazole HO
N
F F
Fluconazole
 
Figure 1. The chemical structures of amphotericin b (Amp b), fluconazole (Flu), and rifampicin (Rif).
Figure 1. The chemical structures of amphotericin b (Amp b), fluconazole (Flu), and rifampicin (Rif). 
As depicted, each of these drugs have unique structural features that can influence membrane integrity.
As depicted, each of these drugs have unique structural features that can influence membrane 
integrity. 
Biosensors 2019, 9, x FOR PEER REVIEW 5 of 12 
Biosensors 2019, 9, 45 5 of 12
Biosensors 2019, 9, x FOR PEER REVIEW 5 of 12 
 
Figure 2. Schematic illustration of the mechanism of drug interaction with biological membranes and 
how its electrochemical response (using a miniature electrode) correlates to cell viability , as capt ured 
Fbiigyu trrhee 2 c..e Slclc choeumnatatitincicgi i ldllluessvttirrcaaetti (oioCnneo ollfoft tmhheeet emre)ec.c hhaannisismmo off drrug iintterraaccttioionn wiitth biiollogiiccall membbrraanneessa and 
hhoow iittss eelleeccttrroocchheemicicaallr reessppoonnssee( (uussiinngg aa miinniiaattuurreee elleeccttrrooddee)) ccoorrrreellaatteesst tooc ceellll vviiaabbiilliittyy,,a assc caapptuturreedd 
3.2. bUbyyV tt-hhVeeI cScee lSlllp cceoocuutrnnottpiinhngogt ddoemevveiitccreey(  (CCSeetlulloldomimese eteterr).). 
3.2. UTVh-Ve ISUSVp/eVcItSro pmhootnoimtoetrriyngS toufd ietshe extracts showed that SEE and SME were very efficient in 
3r.2em. UoVv-iVnIgS  tShpee cbtrioopahcottiovmee ctorym pStouudnieds s, whereas SWE revealed the least as shown in Figure 3. As 
expeTchteedUV/VIS monitoring of the extraThe ,U tVh/eV lIoSw mero anbistoorribnagn coef  vtahleu ee fcrtosmsh tohwe ewdathteart eSxEtEraacntds wSMasE pwreere very efficient in removingxtracts showed that SEE and SsMuEm awbleyr ed uvee rtyo  tehfefi cfaiecnt t thinat  
theiethbeior amctoivste ocfo mthpe ohuynddrso,pwhohbereas SWE revealed the least as shown in Figure 3. As expected, theremoving the bioactive compoiuc ncdoms, pwouhnerdesa cso SuWldE n oret vbeea leexdt rtahcete lde ainstt oa tsh seh aoqwuneo iuns  Fpihgausree , 3o.r  Athse  
lobwioearcatibvseo crbomanpcoeuvnadluse wfreorme nthoet UwVat/eVrIeSx atrcatcivts was presumably due to the fact that either most of theexpected, the lower absorbance value from thee.  Mweatthearn eoxlt raancdt se wthaasn porl,e hsuowmeavbelyr,  dwueer teo m thoer ef aecffti ctiheantt  
hiynd erxotprhacotbinicgc tohme bpioouancdtisvceo muoldlencuotlebse reesxutrlaticntegd inin htioghtheer paqeither most of the hydrophobic compounds could not beeauke oaubssoprhbaasne,coe rinthteenbsiitoiaecst. iNveoncoetmhpelounds extracted into the aqueous phase, eosrs t, hthee  
wmeraejonro Ut UVV/V/IVS IaSbascotirvbea.nMcee twhaanvoelleanngdthetsh waneorle, ihno twhee vsearm, we erraenmgeo r(2e9e0ffi–c2i9e3n t in exbioactive compounds were not UV/VIS active. Methanol and ethanol, hownemv)e frotrra acltli nthget heextbriaocatcst iave, were more efficienntd  
mcoonlefcirumlesedre psureltvinioguisn shtuigdhieesr opfe aDk. arebfsleoxrab—antcheaitn etexntrsaitcitess .hNado nuentihqeulee sws, athveelmenagjotrh UchVa/VIS abin extracting the bioactive molecules resulting in higher peak absorbance intensities. rNaocnteertisstoicrsb ainnceheless, t thhee  
wparveesleenncget hs were in the same range (290–293 nm) for all the extracts and confirmed previous studies ofmajor UV/oVfI Sa natbisooxridbaanntcse,  wphaevneolelnicg tchosm wpeoruen ind st,h ae lskaamloeid rsa,n fglae v(2o9n0o–id2s9,3 c ninmn)a fmora aldlle thhyed eexst,r bacetnsz aennde , 
Da.nrdefl leixgan—int hdaetreivxatrtaivctessh [a1d4,u23n,i2q4u]e.  wavelength characteristics in the presence of antioxidants, phenolicconfirmed previous studies of D. reflexa—that extracts had unique wavelength characteristics in the 
compounds, alkaloids, flavonoids, cinnamaldehydes, benzene, and lignin derivatives [14,23,24].
presence of antioxidants, phenolic compounds, alkaloids, flavonoids, cinnamaldehydes, benzene, 
and lignin derivatives [14,23,24].  
0.3
SWE
SEE
SME
0.25
0.3
SWE
SEE
SME
0.2
0.25
0.15
0.2
0.1
0.15
0.05
0.1
0
0.05
-0.05
0 100 200 300 400 500 600 700 800 900 1000
wavelength (nm)
 
-0.05
Figure 3. 100 200 300 400Figure 3U. VU-VV-IVS IpSro pfirloeffioler tfhoer steheed sweaetder weaxtra 50terc te(0SWE6)0,0seed7e00th 800 900 1000xtract (SWE), saeneodl  eextthraacnto(lS eExEt)r,aacntd (SseEeEd),m aenthda sneoeld 
extract (SME) revealed intense peaks all cenwtearveedleanrgothu n(ndm2)93 nm. The SWE also indicated the least
methanol extract (SME) revealed intense peaks all centered around 293 nm. The SWE a lso indicated 
ptehaek lienatsetn psity, imFigure 3. UeVak-V inISt
pelnysinitgya, low extraction efficiency with water. profile i mfoprl ytihneg  sae loedw  weaxtterar cetixotnra ecftf ic(SieWncEy) ,w siethe dw eatthear.n ol extract (SEE), and seed 
methanol extract (SME) revealed intense peaks all centered around 293 nm. The SWE also indicated 
the least peak intensity, implying a low extraction efficiency with water. 
AbsorbaAnbcseo (rib/ua)nce (i/u)
Biosensors 2019, 9, x FOR PEER REVIEW 6 of 12 
3.3. Cyclic Voltammetry and Cell Viability Studies of S. cerevisiae Cells Treated with Extracts 
To test the electrochemical behavior and redox activity of the extracts, cyclic voltammetry 
analysis was conducted using interdigitated gold electrodes (IDEs), (Metrohm, DropSens). Briefly, 
the IDEs were composed of two interdigitated electrodes with two connection tracks on a glass 
substrate and offered several advantages, such as working with low volumes of samples and 
avoiding tedious polishing of solid electrodes. There were no redox peaks observed from the bare 
electrodes, as shown in Figure 4A (CONT, yellow), however, all the extracts showed quasi-reversible 
oxidation processes in 0.1% DMSO with current values that ranged from 0.10 to 0.18 mA at a scan 
rate of 10 mV/s, as shown in Figure 4A for SWE, red; SME, blue; and SEE, black. The SWE exhibited 
a higher oxidative peak current, which was shifted to the left, probably indicating that most of the 
bioactive compounds were oxidative species compared to those in the SEE and SME extracts. The 
corresponding concentration-dependent cell viability studies were conducted for each extract, and the 
results are shown in Figure 4B. It was observed that SWE (red) demonstrated the most cell death, 
followed by SME (blue) and SEE (black), and the untreated cells CONT (yellow) exhibited the least 
Bceiolsle nvsoiarsb2i0l1i9ty, 9 ,w45ith concentrations up to 30 µg/mL and an incubation time of 8 h. It was noted6 othf 1a2t 
prolonging incubation beyond 8 h resulted in programmed cell death, and the cell counter 
c3o.3n.tCinyuclaicllyV oinltdamicmateetdry earnrodrC melelsVsiaagbeilsi.t y Studies of S. cerevisiae Cells Treated with Extracts
When each extract was tested on S. cerevisiae cell lines, with similar concentrations ranging from 
5 to 3T0o µtge/smt tLh,e deilsetcitnroctc haenmodicica lpbeeahka cvuiorrreantds rwederoex raecctoivridtyedo af tt h1e5 µexgt/rmacLts f,ocry tchliec SvWoltEa mexmtreatcrty (apnuarlpylsei)s, 
wasa sshcoowndnu inct Feidgursein 5gAi.n Atelrsdoi,g ai tnaotetdicegaobldle erleedcotrxo adcetsiv(IiDtyE ws)a, s( Mobesterrovhemd, inD trhoep Spernesse).nBcer ioeffl Sy,MthE e(bIDluEes) 
wanedre ScEoEm (gproeseend) ocfomtwpoairnetde rtdo itghitea utendtreelaetcetrdo cdeellss w(CitOhNtwT,o yceolnlonwec)t aionnd ttrhaec kmseodniuamg lians swshuibcsht rtahtee caenllds 
woffeerree cdulsteuvreerda l(MadEvDa, nrtaedg)e.s T, hsue cmh eatsabwoolirtkeisn ign wthieth mleodwiav aollsuom r ecsoordf esda mpoldesesatn pdeaakv ocuidrirnegnttse dinio tuhes 
psaomliseh cinongcoefnstorlaitdioenle rcatrnogdee as.s Tsheorwenw ienr eFingourreed 5oAx (pMeaEkDs, orbesde)r.v Tedhef rroemsutlhtse wbaerree eilnetcetrropdreetse,da sins hteorwmns 
oinf oFnigeu orre m4Aor(eC bOioNloTg, iycealll opwro),cehsoswese ivnecrl,uadllinthge ienxctrreaacstseds hboiowloegdicqaula msie-rmevberrasnibel pe orxoisdiattyio in tphreo pcersesseesncine 
o0.f1 t%heD eMxtSrOacwtsi wthicthu rSrWenEt ,v eaxlhuiebsitthinagt  rtahneg medosftr oinmfl0u.x1 0oft oio0n.1s8 amt aA 1a5t µags/cmanL reaxtteroafc1t 0comnVce/nst,raastsiohno worn thine 
rFeigleuarsee4 oAf rfoearcStWivEe ,orxeydg; eSnM sEp,ebcliuese ;(RanOdSS) EaEs ,ab rlaescku.ltT ohfe thSeW pEreesxehnibceit eodf tahhe iegxhterraoctxsid. Watiev aelspoe acokrcruerlaretnedt, 
wiohniicch lewaaksasghei fttoe dcetoll tdheealtehft ,bpyr ocobnabdluycitnindgic acetilnl gvtihaabtilmityos sttoufdtihees bwioitahc taivne ecxotmrapcot ucnodnscewnetrreatoixoind oatf iv15e 
µspge/mcieLs aconmd pwairtehd cetollsth ionsceuibnatthede  fSoErE 8a hn,d aSsM shEoewxntr ainc tFsi.gTuhree c5oBr.r eSsWpoEn (dpiunrgpcleo)n creevnteraalteido nc-edlle pdeeantdhe noft 
acebloluvti a5b7i%lit,y wshtuedreieass wSMerEe c(bolnudeu) catned fSoErEe a(gcrheeenxt)r arecct,oarnddedt haebroeustu 3lt1s%a raensdh o22w%n, irneFspigeuctriev4eBly. ,I at tw tahse 
soabmseer vceodncthenattrSaWtioEn(.r eCde)lld demeaothn swtratse dretchoerdmeods tince allnd ienacthre,afosilnlogw oerddebry, SSMEEE (<b lSuMe)Ea n<d SSWEE, (bwlahcikch), 
caonrdretlhaeteudn wtreitahte dthcee lolxs iCdaOtNivTe (pyeealklo wcu)rerxehnitb iinte dthteh esalemaset ocerldlevri, asbuilgitgyeswtiinthg ctohnacte enltercattriocnhseumpictaol 
r3e0sµpgo/nmseLs farnodma tnhein cceullbsa mtioignhttim haevoef r8ehsu. lIttewd ains  nceoltleudlatrh sattrpersosl,o lenagdininggi ntoc uthbae thioignhbeesyt ocneldl d8ehatrhes iunl ttehde 
pinrepsreongcrea mof mtheed Dce. lrlefdleexaat hc,ealln edxtthraeccte (lelscpoeucniatlelryc SoWntEin).u a lly indicated error messages.
(A) (B) 
2000 110
SME
SWE
CONT
1500 SEESEE 100 SME
SWE
CONT
1000 90
500 80
0 70
-500 60
-1000 50
-1500 40
-2000 30
-500 0 500 1000 0 5 10 15 20 25 30 35 40
Voltage(mV)
  
Fiigure 4.. ((A)) Cyclliic Voollttaammeettrryy pprroofiflileeo offt thheee exxtrtraacctstsw wiitthoutt ceellllsss shhoowwiing cchhaarraacctteerrisisttiicc aannoodiic 
siignalls off tthhee mmaainin nantauturarla pl rpordoudcutcs tisn itnhet hseesde wedatwera etexrtreaxcttr (aScWt (ES,W reEd,),r seede)d,  mseetdhamneotlh eaxntroalcet x(StrMacEt, 
b(SluMeE), abnlude )s,eaendd esteheadnoetlh eaxntoralcetx (tSraEcEt, (bSlEaEc,kb),l accokm),pcaormedp awrietdh wthieth btahreeb ealreecterloedcter o(CdeO(NCTO, NyeTl,loywel)l.o (wB). 
C(Bo)nCcoencteranttiroanti-odne-pdeenpdeenndt ecneltl cveiallbviliiatyb islittuydsietusd oife tshoef etxhteraecxttsr oanct sS.o cnerSev. icseiraeev ciseilal elincelsl :l iSnWesE: ,S rWedE; S, rMedE,; 
bSMlueE;, abnlude S; EanEd, bSlaEcEk, ,b cloacmkp, caormedp waritehd twheit hbathre eblaercetreoledcet,r CodOeN, CTO, yNeTllo, ywe.l Rloewp.rRodepurcoibdiulitcyib oilfi tyheo fdtahtae 
wdatsa awnaslyazneadly uzseidngu strinipglitcraipteli mcaeteasmuereamsuernemts.e nts.
When each extract was tested on S. cerevisiae cell lines, with similar concentrations ranging from
5 to 30 µg/mL, distinct anodic peak currents were recorded at 15 µg/mL for the SWE extract (purple),
as shown in Figure 5A. Also, a noticeable redox activity was observed in the presence of SME (blue) and
SEE (green) compared to the untreated cells (CONT, yellow) and the medium in which the cells were
cultured (MED, red). The metabolites in the media also recorded modest peak currents in the same
concentration range as shown in Figure 5A (MED, red). The results were interpreted in terms of one or
more biological processes including increased biological membrane porosity in the presence of the
extracts with SWE, exhibiting the most influx of ions at a 15 µg/mL extract concentration or the release
of reactive oxygen species (ROS) as a result of the presence of the extracts. We also correlated ionic
leakage to cell death by conducting cell viability studies with an extract concentration of 15 µg/mL
and with cells incubated for 8 h, as shown in Figure 5B. SWE (purple) revealed cell death of about
57%, whereas SME (blue) and SEE (green) recorded about 31% and 22%, respectively, at the same
concentration. Cell death was recorded in an increasing order, SEE < SME < SWE, which correlated
with the oxidative peak current in the same order, suggesting that electrochemical responses from
Current (mA)
Current (mA)
Biosensors 2019, 9, 45 7 of 12
tBhieosecneslolrss m201ig9,h 9t, hx aFOveR PreEsEuRl tReEdViIEnWc ellular stress, leading to the highest cell death in the presence o7f othf 1e2 
D. reflexa cell extract (especially SWE).
 
Figure 5. (A) Concentration-dependent profile of the change in anodic current as a function of extract
Figure 5. (A) Concentration-dependent profile  of the change in anodic current as a function of extract 
concentration after 8 h of extract administration (SWE, purple; SME, blue; and SEE, green) compared
concentration after 8 h of extract administration (SWE, purple ; SME, blue; and SEE, green) compared 
to untreated cells (CONT, yellow) and the medium in which the cells were cultured (MED, red).
to untreated cells (CONT, yellow) and the medium in which the cells were cultured (MED, red). (B) 
(B) Percent cell viability at 15µg/mL extract concentration with data recorded from 20 min to 8 h using
Percent cell viability at 15µg/mL extract concentration with data recorded from 20 min to 8 h using 
trypan blue as a staining dye. SWE, purple; SME, blue; and SEE, green were compared to the untreated
trypan blue as a staining dye. SWE, purple; SME, blue ; and SEE, green were compared to the 
cells (CONT, yellow) after 8 h. Reproducibility of the data was analyzed using triplicate measurements.
untreated cells (CONT, yellow) after 8 h. Reproducibility of the data was analyzed using triplicate  
3.4. CmycelaicsuVroelmtaemnmtse. try Studies of S. cerevisiae in the Presence of Antifungal Drugs
3.4. CTyhcelice xVtorlatcatmdmaettray wSatusdcieosm opf aSr. ecderetvoisAiame pin bthbe ePcraeusesnecet hoef aAnnttiiffuunnggaall Dabruilgisty of the drug to cause
cell death has been linked to cellular stress and ionic leakage, with numerous electrochemical
studiTeshes uegxgtreasctitn dgataa cwoarrse claotmiopnarbeedtw toe Aenmmp ebm bbercaanuesep tehrem aenatbifiulintygaolf aiboinlistyt ooft thhee ddrruugg mtoe ccahuasnei scmell 
odfeaactthio hna[s2 4b,e2e5n]. lAinsksehdo wton cienllFuilgaurr set6rAes,sc ayncldic iovnolitca mleamkeatgrye, mweiatshu nreummeenrtosuosf etrleeactterdoccheelmls iwcaitlh sdturudgiess 
wsuegregecsotminpga ar ecdortroetlhateiocnel lbseatwloeneen.  Ammempbbra(bnleu pe)ertrmeaeatebdilicteyll osfw ioitnhs ctoon tcheen dtrrautigo nmsercahnagniinsgmf roof mac5titoon 
3[024µ, g2/5]m. ALss shhoowwend ienl eFvigauterde 6aAno, dcyicclriecs vpoolntasems,minetdriyc amtienagsuiorenmicelnetask oafg terefarotemd caelplso rwouitshm dreumgbs rwaneere,  
acsomprpevairoeuds tlyo dtheme ocenlslstr aatleodne[.2 4A,2m5]p. Sbi m(billuare)t rteraetamteedn tcweliltsh wFliuth( pcounrpcelen)tsrhaotiwonesd rnaongdirnagm fartoicmc h5a tnog e3s0 
iµnga/mnoLd ischpoewakedc uerlreevnatteind tahneopdriecs ernescpeoonfstehse, dinrudgic,aatnindgt hioisniocb sleearkvaagtieo nfrwomas ath peosraomues wmheemnbtrhaencee,l lass 
wpreerve itorueasltyed dewmitohnsRtirfa(tgerde [e2n4), a2f5t]e.r Seimigihlat rh toruearstmofendtr wugitehx Fplous (upruer, pcloem) sphaorwededto ntoh derAammaptibc cchuarrnegnets 
riens apnoondseic wpeitahki ncuthrreenmta inrg tihne opfrsetsaetnisctei coafl tehrer odrr.ugP,r aionrdt tohtihs eobtsreearvtmateinont  owfatsh tehcee slalsmwe withhethne thder cueglsls,  
cwycelriec tvroelatatemdm weittrhy Rainf a(lgyrseisenin) daifctaerte edigthhta thAoumrps oBf wdarsugn oetxrpeodsouxrea,c tciovme,pyaerteidts tmo ethche aAnmismp bof caucrtrioennt 
croeuspldoncrseea wteitphoirne tshine mthaercgeinll uolfa srtamtiesmtibcaral nererotor.e Pnrhiaonr ctoe tthhee tirneflautmx oenf ito onf sthine caenldls owuitthof tthhee dmruegms,b cryacnleic,  
ivnodlitcaamtimngetmrye manbarlaynseis- mineddiciaatteedd tehffaetc Atsm(dpa Bta wnaost nsohto rwedno).x Tahctuisv,ei, tyweta istsc monefichrmaneidsmth oaft aFcltuioann cdoRulidf  
wcreeraetne optomreesm inb rtahnee c-melleudliacra tmedem, abnrda,nteh etroe efonrhea, nmceem thber ainneflupxe romf eiaotniosn ino fainodn sowuta sofv ethrye mmienmimbraalnaes, 
rienvdeiaclaetdining tmheemdabtraa. nTeh-emseedobiasteerdv aetfifoenctssa (tdteasttae dntoot esahroliwerns)t.u Tdhieusst,h iat twthaesm coecnhfiarnmisemd othf aactt Fiolun oafntdh eRseif 
dwruerges nwoats mthermoubgrahndei-fmfeerednictaptaetdh, wanayds, ,thbuertetfhoerye,c mouelmdbsrtiallnde epmeromnsetartaitoens oomf ieonlesv welaosf veelercyt rmocinhiemmailc aals 
rreesvpeoanlesdes i[n2 6th,2e7 ]d.ata. These observations attested to earlier studies that the mechanism of action of 
theseT hderusegcso nwdaps htahsreouwgahs dtoiffceorrernetla tpeatthhewealyecst, rboucht etmheicya lcroeuslpdo nssteillo df eemacohndstrruagtet osocemlle vlieavbeilli toy,f 
aesleschtroowcnheimn iFciaglu rreesp6oBn. sTehse [2c6el,2l 7v]i.a b ility percentage was recorded using trypan blue as the staining
agentTfhroe msec2o0nmd ipnhtaos8e wh ians ttho ecoprrreesleantec ethoef eelaecchtrdocruhgem, aiscawl raessppeornfsoer mofe edawchi tdhrtuhge teox cterallc vtsi.aTbhileityce, alls 
vsihaobwilinty inre Fsiuglutsrea 6ftBe.r T2h0em ceinll dviidabniolittysh poewrceandtaragset iwc achs arencgoerdinedc eulsl idnega ttrhywpahne nblturee aatse tdhwe sitthaitnhiengd arugegnst. 
Hfroowme 2v0e mr, itnh etroe 8w ha isn athder parsetiscenchcea nogf eeaicnhc derlluvgi,a absi lwitayso pfearbfoorumt e6d4 %wiitnh tthhee epxrtersaecntcse. TohfeF cleull (vpiuabrpilliety)  
arfetseurletisg ahfttehro 2u0r ms. iAn mdipd bno(bt sluheo)wsh ao dwraedsticce cllhdaneagteh ino fcealbl odueat t3h5 %w,hwenh terreeaatseRd iwf (igthre tehne) drreucgosrd. Hedowabevouert, 
1t6h%erec ewllads eaa dthraastticco cnhcaenngtrea itnio cnesll ovfia1b5ilµitgy/ mof La.bPoruetv 6io4u%s isnt uthdeie psroensenFclue oinf dFilcua t(epdurtphlaet) caefltlerd eeiagthht 
whoaus rnso. tAmmepm bb r(banluee-)m sehdoiwateedd ,caelnl ddewaaths, otfh earbeofourte 3,5u%se, dwahseraeacso nRtirfo (lgfrueenng)i srteactiocrdderdu ga,bwouhte 1re6a%s Rceilfl 
sdeeravtehd aats caonnacnentitbraacttieornias lodf r1u5g µthga/mt hLa. sPnroevaniotuifsu nstguadl iperso opne rtFileus . iIntdwicaast,etdh etrheafot rcee,llc odnecaltuhd ewdafsr onmot 
tmhiesmstburdaynet-hmatedalitaetreadt,i oannsdo wf athse, tehnedreofgoerne,o uussemd eams ab rcaonnetrdoul efutnogtihsetaptirce sdernucge, owfhAemrepasb Rmif isgehrtvheadv aes 
raensu alntetdibianctaenrieanl hdaruncge tdhaintfl huaxs nofo iaonntsi,fuanndgatlh pisrocoprerretliaetse. dItt wo tahse, tihnecrreeafosreed, ccoelnlcdlueadtehdt fhraotmh atshaislr setauddyy 
btheeant adlotecruamtioennste odf t[h22e, 2en8]d.oAgesnporuesv imouems fibnrdanineg dsuhea tvoe tihned picraetseedn,cme oafn Ay mbipo lbog miciaglhmt heamvber raenseusltheadv ien 
an enhanced influx of ions, and this correlated to the increased cell death that has already been 
documented [22,28]. As previous findings have indicated, many biological membranes have 
electrochemical characteristics, which is important for the generation of electron transfer in living 
systems [29–32]. Thus, endogenous chemical imbalances and an increased influx of drugs in the cell 
Biosensors 2019, 9, 45 8 of 12
electrochemical characteristics, which is important for the generation of electron transfer in living
Biosensors 2019, 9, x FOR PEER REVIEW 8 of 12 
systems [29–32]. Thus, endogenous chemical imbalances and an increased influx of drugs in the cell
ccaann rreessuulltt iinn cceelllluullaarr ssttrreessss,, lleeaaddiinngg ttoo cceellll ddeeaatthh.. AAnnyy nneeww mmoolleeccuullee oorr ddrruugg eennttiittyy wwiitthh tthhee aabbiilliittyy ttoo 
iinnccrreeaassee mmeemmbbrraannee ppeerrmmeeaabbiilliittyyo off iioonnss iiss lliikkeellyy ttoo iinnccrreeaassee cceelllluullaarr ssttrreessss.. WWhhiillee iitt iiss aacckknnoowwlleeddggeedd 
tthhaatt cceellll ddeeaatthh mmaayy sshhooww ddiiffffeerreenntt mmeecchhaanniissmm ooff iinnhhiibbiittiioonn,, iitt iiss eexxppeecctteedd ffrroomm tthhiiss ssttuuddyy tthhaattp pllaanntt 
eexxttrraaccttss,,o orri siosloaltaetdedb iobaioctaicvteivcoe mcopmoupnodusnfdrso mfrpomlan ptsl,acnatns,b ceauns ebde toussetudd tyo cestlluudlayr csetrlelusslaarn sdtrceosrsre alantde 
tchoerirrealactteiv tihtyeitro accetilvl idteya ttoh cueslli ndgeabtoht huseilnegc tbroocthhe emleicctarloacnhdemceicllavl aianbdil citeylls vtuiadbieilsit[y33 s,t3u4d].ies  [33,34].  
0.3 100
Amp b
Rif
Flu 90
Cont
Med
0.25 80
70
0.2 60
50
0.15 40
30
0.1 20
10
0.05 0
-5 0 5 10 15 20 25 30 35 20 60 120 180 240 480
Time(Mins)
  
(A) (B) 
FFiiggurree 66.. ((A)) Cooncceennttrraattiioonn--ddeeppeenndeentt prroofifillee ooff tthee  cchaanggee iin aanoodiicc ccurrrreentt aass aa ffunccttiioon ooff drrugg 
conceennttrraatitoionn( (aammpp b,, bllue;; Riiff,, greeenn;; Fllu,, purplle;; unttreaatted celllls,, CONT,, yellllow;; and mediium,, MED,, 
red)) afftterr8 8 h off drug admiiniisttrraattiioonn.. ((B)) Perrcceennttc ceellllv viiaabbiliilittyya att1 155 µg/mL exttractt concceennttrraattiioonn wiitth 
datta rreccoorrddeeddf frroom2 200 miin tto 8 h usiing ttrrypan bllue ass a ssttaaininiinngg dyee.. Reepprroodduucciibbiilliitty off tthe datta was 
anallyzeedd usiing ttrriipllicattee meeaassuurreemeenntsts. . 
33..55.. DDiissccuussssioionn 
TThheerree aarree nnuummereorouus srerdeodxo mx emdieadtioartso trhsatth aarte uarseedu tsoe deitthoere sitthuedry scteulld ryedcoexll arcetdivoixtya ocrt diveivtyeloopr 
dbieovseelnosporbsi ofosre nmsaonrsy fboirolomgaicnayl sbyiostloemgisc.a Flosry esxtaemmsp.le, FSo. rceerexvaimsiape leh,aSs .secvereervaisl iraeedhoxa scesnetveerrsa, lsurechd oaxs 
c[Feen(tCeNrs), such as [Fe(CN) ]
3−
]3−/[Fe(CN) /[Fe(CN) ]
4− and NAD(P)H/NAD(P)+, which can be targeted by
6 6]4− and6 NAD(P)H/NA6 D(P)+, which can be targeted by hydrophilic/hydrophobic 
hmyodlerocuplheisl,i ca/sh eyxdternospihvoeblyic dmiscoulescsuedle ps,raevs ieoxutselnys bivye Rlyawdissocuns este adl.p [r3e5v,3io6u].s Ilny abdydRitaiowns, oelnecettraolc.h[e3m5,i3c6a]l. 
Imnoandidtoitriionng, cealenc tbroe cchoenmdiuccatlemd ownihtoenri nregaccatnivbee ocxoyngdeunc stpedecwiehse anrree raeclteiavseeodx aysg ean respsueclti eosf adrreurge-leinasdeudceads  
aacrteisounl t[o3f2,d3r7u–g4-0in].d Ruecaecdtiavcet iooxny[g3e2n,3 7s–p4e0c]i.eRs eiasc taiv teeromxy guesneds pteoc idesesicsraibteer omxyugseend tsopdeceisecsr,i biencolxuydgineng 
sspuepceireos,xiindcel uadniinogn sruapdeiro
•−
caxl i(dOe a•−n) ioanndra hdyicdarlo(Oge2n p)earnodxihdyed (rHogOen),p earnodx idthee(yH c2aOn2 )c,aaunsde tchyetyoctaonxicca au2 2 2 nsde 
caynttoimtoxicircoabnidala enffteimctsic irno mbiaolste fofregcatsniinsmmso. sTthoursg,a tnhiesrme sa.rTe hatu lse,athste trheraeree maat ilnea mstetchhraeneimsmaisn omf dercuhga nacistmions  
oinfcdlurudginagc,t ibounti nncoltu ldiminigt,ebdu tton, motelmimbitreadneto d,empeomlabrirzaanteiodne pleoaldarinizga ttioo nanle iandfilnugx toof aionnins fl(ausx ionf tihoen sca(asse ionf 
tAhme cpa bse), otfarAgmetpinbg) o, ft arregdeotxin cgenotferresd, oaxndce/onrt etrhse, raenldea/soer otfh Re OreSle ians eS.o cfeRreOvisSiaien S[2.8c,e3r5ev,3is6i,a4e1[]2. 8A,3m5p,3 b6, 4h1a]s. 
Abemenp busheads btoe etnreuaste fdutnogtarle aint ffeucntgioanl ,i nafnecdt iothne,  amnedcthhaenmisemch tahnriosumghth wrohuigchh wthheic ahntthiefuanngtaiflu dnrguagl  dkriullgs 
kinilflescitnefde ccteeldls cise lwlseilsl-wcheallr-cahctaerraiczteedri uzesidnug sbinotghb boitohpbhiyospichayls aicnadl manidcrmobiciorolobgioicloagl ticeachl tneicqhuneisq [u2e2s,2[25]2., 2T5h]e. 
Tanhteifaunntgifauln dgraulgd bruingdbs itnod esrtgoosetregrooslt earnodl faonrdmfso prmorsesp oarte tshaet ctehlle mceelml mbreamnber, acnaue,sicnagu stihneg lothsse olof sisonosf  
iaonnds alenaddilneagd tino gdteopdoleaproizlaartiizoant ioofn tohfet hmeemmebmrabnraen [e1[31,231,2]1. ]T. hTihsi smmecehchananisimsm ccaann ccaauussee aann eennhhaanncceedd 
ooxxiiddaattiioonn ppootteennttiiaall,, wwhhicichhc caannb beec acpaptuturerdedth trhoruoguhghel eelcetcrtorcohcehmemicaicladle dteecteticotnio,na,s aaslr aelardeyadoyb soebrsveerdveind 
oinu or ustru sdtiuedsiaess awse wll ealsl satsu sdtiuedsiferso fmroomth oetrhgerro gurposup[2s5 [,3265],.36].  
IInn tthhee ccaassee ooff tthhee ttwwoo ootthheerr mmeecchhaanniissmmss,, aann eennhhaanncceedd aannooddiicc rreessppoonnssee ccaann rreessuulltt ffrroomm eeiitthheerr 
ttaarrggeettiinngg rreeddooxx mmeeddiiaattoorrc ceenntetersrso orrg geenneerartaitnigngo noeneo rorm moroerRe ORSOsSp sepceiecsieass aas rae sruelstuoltf odfr udgrubgin bdinindgi,nags, 
dase pdiecpteicdtebde lboewlo:w: 
NADPH ←→ NADP+, (1)
H+ + 2e−
NADP−H→       ⟷    +     NAD−P+, 
(1) 
H2O2 O2 (gH) + +2 2He- (aq) + 2e . (2)
H2O2 ⟶ O2 (g) + 2H+ (aq) + 2e−. (2) 
Whereas the reduction peaks seemed to deviate from a typical redox reaction, the research 
suggested that the effect of Amp b (35%) or the plant extracts  (57%) on S. cerevisiae viability, which 
corresponded to the quasi-reversible oxidation process, inevitably supported a general claim that 
Current (mA)
Cell Viability(%)
Biosensors 2019, 9, 45 9 of 12
Whereas the reduction peaks seemed to deviate from a typical redox reaction, the research
suggested that the effect of Amp b (35%) or the plant extracts (57%) on S. cerevisiae viability,
which corresponded to the quasi-reversible oxidation process, inevitably supported a general claim
that Amp b (57%) and the plant extracts (SWE, SME, SEE at 57%, 31%, and 22%, respectively) behave
similarly and have the ability to create ionic pores in fungal cells that leads to their death. Our data
was reproducible because it used triplicate measurements from the same extract or from different
extracts of the same plant, with error margins of less than 5% confidence. The selectivity towards the
extracts was validated using the non-membrane-targeted antimicrobial drug, Rif, with the extracts,
revealing up to 57% antifungal activity compared to 16% of the antimicrobial drug. These studies
provide a sensitive sensing method that can electrochemically detect plant extracts, causing either
depolarization of membranes, accumulation of ROS, or targeting redox centers in S. cerevisiae cell
cultures. Whereas the fabrication of microelectrodes for electrochemical studies has been going on
for one or two decades in various fields, there is limited application in the natural product field.
The limited evidence of the application of electrochemical methods in natural products screening,
as shown in Table 1, might be due to the high cost of operation [42]. As shown in Table 1, our method
offers a simple and straightforward screening platform to select active natural product pools in a fast
and robust fashion prior to undertaking further validation studies [43,44].
Table 1. Comparing the various methods for screening natural product activity towards molecular targets.
Method Strategy Sample Size Robustness IncubationTime (h) Disadvantages
Uses Interdigitated
The current Method electrode and microliters Fast andSensitive 0.3–8
Preliminary
Cellometer Target not known
Fluorescence Based on staining
techniques and counter milliliters
Complex long phototoxic-unclear
staining architecture images [44]
High Performance
Liquid
Chromatography A separation
-Electrochemical technique microliters
selective and
sensitive Relatively fast Expensive [44]
Detection
(HPLC-ECD)
Structural Activity nuclear magnetic target-directed Focuses on hitRelationship by NMR resonance microliters Short validation
(SAR by NMR) (NMR)-based drug research studies [43]
Agar plates are
Diffusion Methods inoculated and large Simplicity andcompared to low cost 16–24 Inaccuracies [43]
standard
4. Conclusions
At a concentration of 15 µg/mL, S. cerevisiae cell lines revealed unique oxidation peak responses
at 0.34, 0.25, and 0.23 in the presence of SWE, SME, and SEE of the D. reflexa extracts, respectively,
correlating to cell death of 57%, 31%, and 22% in that order. The results were comparable to Amp b
on S. cerevisiae cell death, where the highest oxidation peak current was directly related to inhibitory
effects. Thus, one or many bioactive compounds in D. reflexa might induce cell death through similar
mechanisms as Amp b. Future work will focus on the individual bioactive compounds from the water
extracts of D. reflexa seeds to validate their potential therapeutic application. In conclusion, the current
studies have highlighted the robustness of electrochemical detection in monitoring cell death using
microliter volumes of sample.
Biosensors 2019, 9, 45 10 of 12
Author Contributions: E.K.T. and P.K.A. conceived and designed the experiments; A.B.Y., I.I. and S.B.
performed the experiments; E.K.T., B.O.A., E.J.F. and S.K.K. analyzed the data; P.K.A., I.I. and S.B. contributed
reagents/materials/analysis tools; E.K.T. and P.K.A. wrote the paper.
Funding: The work received support from Wellcome Trust (WACCBIP DELTAS grant; 107755/Z/15/Z (PKA and
EKT), and the World Bank Africa Centres of Excellence project ACE02-WACCBIP. We also thank Alfred Ntiamoah
for assisting in data processing using MATLAB.
Conflicts of Interest: The authors declare no conflict of interest.
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